Certain chemical entities, compositions, and methods

ABSTRACT

Certain substituted urea derivatives selectively modulate the cardiac sarcomere, for example by potentiating cardiac myosin, and are useful in the treatment of systolic heart failure including congestive heart failure.

This application is a divisional of U.S. patent application Ser. No.12/772,872, filed on May 3, 2010, now U.S. Pat. No. 8,410,108, which isa divisional of U.S. patent application Ser. No. 11/639,390, filed onDec. 13, 2006, now U.S. Pat. No. 7,718,657, which claims the benefit ofU.S. Provisional Patent Application No. 60/751,118, filed on Dec. 16,2005, all of which are incorporated herein by reference.

The invention relates to certain substituted urea derivatives,particularly to certain chemical entities that selectively modulate thecardiac sarcomere, and specifically to certain chemical entities,pharmaceutical compositions and methods for treating heart disease.

The “sarcomere” is an elegantly organized cellular structure found incardiac and skeletal muscle made up of interdigitating thin and thickfilaments; it comprises nearly 60% of cardiac cell volume. The thickfilaments are composed of “myosin,” the protein responsible fortransducing chemical energy (ATP hydrolysis) into force and directedmovement. Myosin and its functionally related cousins are called motorproteins. The thin filaments are composed of a complex of proteins. Oneof these proteins, “actin” (a filamentous polymer) is the substrate uponwhich myosin pulls during force generation. Bound to actin are a set ofregulatory proteins, the “troponin complex” and “tropomyosin,” whichmake the actin-myosin interaction dependent on changes in intracellularCa²⁺ levels. With each heartbeat, Ca²⁺ levels rise and fall, initiatingcardiac muscle contraction and then cardiac muscle relaxation. Each ofthe components of the sarcomere contributes to its contractile response.

Myosin is the most extensively studied of all the motor proteins. Of thethirteen distinct classes of myosin in human cells, the myosin-II classis responsible for contraction of skeletal, cardiac, and smooth muscle.This class of myosin is significantly different in amino acidcomposition and in overall structure from myosin in the other twelvedistinct classes. Myosin-II consists of two globular head domains linkedtogether by a long alpha-helical coiled-coiled tail that assembles withother myosin-IIs to form the core of the sarcomere's thick filament. Theglobular heads have a catalytic domain where the actin binding and ATPfunctions of myosin take place. Once bound to an actin filament, therelease of phosphate (cf. ATP to ADP) leads to a change in structuralconformation of the catalytic domain that in turn alters the orientationof the light-chain binding lever arm domain that extends from theglobular head; this movement is termed the powerstroke. This change inorientation of the myosin head in relationship to actin causes the thickfilament of which it is a part to move with respect to the thin actinfilament to which it is bound. Un-binding of the globular head from theactin filament (also Ca²⁺ modulated) coupled with return of thecatalytic domain and light chain to their startingconformation/orientation completes the contraction and relaxation cycle.

Mammalian heart muscle consists of two forms of cardiac myosin, alphaand beta, and they are well characterized. The beta form is thepredominant form (>90 percent) in adult human cardiac muscle. Both havebeen observed to be regulated in human heart failure conditions at bothtranscriptional and translational levels, with the alpha form beingdown-regulated in heart failure.

The sequences of all of the human skeletal, cardiac, and smooth musclemyosins have been determined. While the cardiac alpha and beta myosinsare very similar (93% identity), they are both considerably differentfrom human smooth muscle (42% identity) and more closely related toskeletal myosins (80% identity). Conveniently, cardiac muscle myosinsare incredibly conserved across mammalian species. For example, bothalpha and beta cardiac myosins are >96% conserved between humans andrats, and the available 250-residue sequence of porcine cardiac betamyosin is 100% conserved with the corresponding human cardiac betamyosin sequence. Such sequence conservation contributes to thepredictability of studying myosin based therapeutics in animal basedmodels of heart failure.

The components of the cardiac sarcomere present targets for thetreatment of heart failure, for example by increasing contractility orfacilitating complete relaxation to modulate systolic and diastolicfunction, respectively.

Congestive heart failure (“CHF”) is not a specific disease, but rather aconstellation of signs and symptoms, all of which are caused by aninability of the heart to adequately respond to exertion by increasingcardiac output. The dominant pathophysiology associated with CHF issystolic dysfunction, an impairment of cardiac contractility (with aconsequent reduction in the amount of blood ejected with eachheartbeat). Systolic dysfunction with compensatory dilation of theventricular cavities results in the most common form of heart failure,“dilated cardiomyopathy,” which is often considered to be one in thesame as CHF. The counterpoint to systolic dysfunction is diastolicdysfunction, an impairment of the ability to fill the ventricles withblood, which can also result in heart failure even with preserved leftventricular function. Congestive heart failure is ultimately associatedwith improper function of the cardiac myocyte itself, involving adecrease in its ability to contract and relax.

Many of the same underlying conditions can give rise to systolic and/ordiastolic dysfunction, such as atherosclerosis, hypertension, viralinfection, valvular dysfunction, and genetic disorders. Patients withthese conditions typically present with the same classical symptoms:shortness of breath, edema and overwhelming fatigue. In approximatelyhalf of the patients with dilated cardiomyopathy, the cause of theirheart dysfunction is ischemic heart disease due to coronaryatherosclerosis. These patients have had either a single myocardialinfarction or multiple myocardial infarctions; here, the consequentscarring and remodeling results in the development of a dilated andhypocontractile heart. At times the causative agent cannot beidentified, so the disease is referred to as “idiopathic dilatedcardiomyopathy.” Irrespective of ischemic or other origin, patients withdilated cardiomyopathy share an abysmal prognosis, excessive morbidityand high mortality.

The prevalence of CHF has grown to epidemic proportions as thepopulation ages and as cardiologists have become more successful atreducing mortality from ischemic heart disease, the most common preludeto CHF. Roughly 4.6 million people in the United States have beendiagnosed with CHF; the incidence of such diagnosis is approaching 10per 1000 after 65 years of age. Hospitalization for CHF is usually theresult of inadequate outpatient therapy. Hospital discharges for CHFrose from 377,000 (in 1979) to 970,000 (in 2002) making CHF the mostcommon discharge diagnosis in people age 65 and over. The five-yearmortality from CHF approaches 50%. Hence, while therapies for heartdisease have greatly improved and life expectancies have extended overthe last several years, new and better therapies continue to be sought,for example, for CHF.

“Acute” congestive heart failure (also known as acute “decompensated”heart failure) involves a precipitous drop in cardiac function resultingfrom a variety of causes. For example in a patient who already hascongestive heart failure, a new myocardial infarction, discontinuationof medications, and dietary indiscretions may all lead to accumulationof edema fluid and metabolic insufficiency even in the resting state. Atherapeutic agent that increases cardiac function during such an acuteepisode could assist in relieving this metabolic insufficiency andspeeding the removal of edema, facilitating the return to the morestable “compensated” congestive heart failure state. Patients with veryadvanced congestive heart failure particularly those at the end stage ofthe disease also could benefit from a therapeutic agent that increasescardiac function, for example, for stabilization while waiting for aheart transplant. Other potential benefits could be provided to patientscoming off a bypass pump, for example, by administration of an agentthat assists the stopped or slowed heart in resuming normal function.Patients who have diastolic dysfunction (insufficient relaxation of theheart muscle) could benefit from a therapeutic agent that modulatesrelaxation.

Inotropes are drugs that increase the contractile ability of the heart.As a group, all current inotropes have failed to meet the gold standardfor heart failure therapy, i.e., to prolong patient survival. Inaddition, current agents are poorly selective for cardiac tissue, inpart leading to recognized adverse effects that limit their use. Despitethis fact, intravenous inotropes continue to be widely used in acuteheart failure (e.g., to allow for reinstitution of oral medications orto bridge patients to heart transplantation) whereas in chronic heartfailure, orally given digoxin is used as an inotrope to relieve patientsymptoms, improve the quality of life, and reduce hospital admissions.

Given the limitations of current agents, new approaches are needed toimprove cardiac function in congestive heart failure. The most recentlyapproved short-term intravenous agent, milrinone, is now nearly fifteenyears old. The only available oral drug, digoxin, is over 200 hundredyears old. There remains a great need for agents that exploit newmechanisms of action and may have better outcomes in terms of relief ofsymptoms, safety, and patient mortality, both short-term and long-term.New agents with an improved therapeutic index over current agents willprovide a means to achieve these clinical outcomes.

Current inotropic therapies improve contractility by increasing thecalcium transient via the adenylyl cyclase pathway, or by delaying cAMPdegradation through inhibition of phosphodiesterase (PDE), which can bedetrimental to patients with heart failure.

Given the limitations of current agents, new approaches are needed toimprove cardiac function in congestive heart failure. The most recentlyapproved short-term intravenous agent, milrinone, is more than fifteenyears old. The only available oral drug, digoxin, is over 200 hundredyears old. There remains a great need for agents that exploit newmechanisms of action and may have better outcomes in terms of relief ofsymptoms, safety, and patient mortality, both short-term and long-term.New agents with an improved therapeutic index over current agents willprovide a means to achieve these clinical outcomes.

The selectivity of agents directed at the cardiac sarcomere (forexample, by targeting cardiac beta myosin) has been identified as animportant means to achieve this improved therapeutic index. The presentinvention provides such agents (particularly sarcomere activatingagents) and methods for their identification and use.

Another approach may be to directly activate cardiac myosin withoutchanging the calcium transient to improving cardiac contractility. Thepresent invention provides such agents (particularly myosin activatingagents) and methods for their identification and use.

The present invention provides chemical entities, pharmaceuticalcompositions and methods for the treatment of heart failure includingCHF, particularly systolic heart failure. The compositions are selectivemodulators of the cardiac sarcomere, for example, potentiating cardiacmyosin.

The present invention provides at least one chemical entity chosen fromcompounds of Formula I

and pharmaceutically acceptable salts, solvates, chelates, non-covalentcomplexes, prodrugs, and mixtures thereof,

where one of Z¹ and Z² is —NR¹⁵C(O)NR¹⁶R⁴ and the other of Z¹ and Z² isR³;

R⁴ is chosen from optionally substituted aryl, optionally substitutedaralkyl; optionally substituted cycloalkyl, optionally substitutedheteroaryl, optionally substituted heteroaralkyl and optionallysubstituted heterocycloalkyl;

R³ is chosen from hydrogen, halo, cyano, hydroxyl, optionallysubstituted alkyl, optionally substituted heterocycloalkyl, andoptionally substituted heteroaryl;

R¹ and R² are independently chosen from hydrogen, halo, cyano,optionally substituted alkyl, optionally substituted heterocycloalkyl,and optionally substituted heteroaryl;

R¹⁵ and R¹⁶ are independently chosen from hydrogen, and optionallysubstituted alkyl;

W¹ is chosen from N and C;

A is chosen from cycloalkyl, heterocycloalkyl, aryl, and heteroarylgroups having from 5 to 7 ring atoms including the atoms shared with the6 membered aromatic ring containing W¹;

R⁵ is chosen from optionally substituted alkyl, optionally substitutedamino, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted cycloalkyl and optionally substitutedheterocycloalkyl;

p is 0 or 1;

R⁶ is chosen from optionally substituted alkyl, and halo;

L is chosen from a bond, optionally substituted lower alkylene, —O—,—O-(optionally substituted lower alkylene)-, -(optionally substitutedlower alkylene)-O—, —S—, —S-(optionally substituted lower alkylene)-,-(optionally substituted lower alkylene)-S—, —SO₂—, —SO₂-(optionallysubstituted lower alkylene)-, and -(optionally substituted loweralkylene)-SO₂—

provided that if R⁵ is amino or if R⁵ is heteroaryl or heterocycloalkylwith a heteroatom bonded to L, then L is not —O—, —S—, —O-alkyl, or—S-alkyl.

Also provided are pharmaceutical compositions comprising apharmaceutically acceptable excipient or adjuvant and at least onechemical entity as described herein.

Also provided are packaged pharmaceutical compositions, comprising apharmaceutical composition as described herein and instructions forusing the composition to treat a patient suffering from a heart disease.

Also provided are methods of treating heart disease in a mammal whichmethod comprises administering to a mammal in need thereof atherapeutically effective amount of at least one chemical entity asdescribed herein.

Also provided are methods for modulating the cardiac sarcomere in amammal which method comprises administering to a mammal in need thereofa therapeutically effective amount of at least one chemical entity asdescribed herein.

Also provided are methods for potentiating cardiac myosin in a mammalwhich method comprises administering to a mammal in need thereof atherapeutically effective amount of at least one chemical entity asdescribed herein.

In certain embodiments, the present invention provides methods ofscreening for chemical entities that will bind to myosin (for example,myosin II or β myosin), for example chemical entities that will displaceor compete with the binding of at least one chemical entity as describedherein. The methods comprise combining an optionally-labeled chemicalentity as described herein, myosin, and at least one candidate agent anddetermining the binding of the candidate agent to myosin.

In certain embodiments, the invention provides methods of screening formodulators of the activity of myosin. The methods comprise combining achemical entity as described herein, myosin, and at least one candidateagent and determining the effect of the candidate agent on the activityof myosin.

Other embodiments will be apparent to those skilled in the art from thefollowing detailed description.

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

As used herein, when any variable occurs more than one time in achemical formula, its definition on each occurrence is independent ofits definition at every other occurrence. In accordance with the usualmeaning of “a” and “the” in patents, reference, for example, to “a”kinase or “the” kinase is inclusive of one or more kinases.

Formula I includes all subformulae thereof. For example Formula Iincludes compounds of Formula Ia, Ib, II, etc.

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example, —CONH₂ isattached through the carbon atom.

By “optional” or “optionally” is meant that the subsequently describedevent or circumstance may or may not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “optionally substituted alkyl”encompasses both “alkyl” and “substituted alkyl” as defined below. Itwill be understood by those skilled in the art, with respect to anygroup containing one or more substituents, that such groups are notintended to introduce any substitution or substitution patterns that aresterically impractical, synthetically non-feasible and/or inherentlyunstable.

“Alkyl” encompasses straight chain and branched chain having theindicated number of carbon atoms, usually from 1 to 20 carbon atoms, forexample 1 to 8 carbon atoms, such as 1 to 6 carbon atoms. For exampleC₁-C₆ alkyl encompasses both straight and branched chain alkyl of from 1to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl,isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, and thelike. Alkylene is another subset of alkyl, referring to the sameresidues as alkyl, but having two points of attachment. Alkylene groupswill usually have from 2 to 20 carbon atoms, for example 2 to 8 carbonatoms, such as from 2 to 6 carbon atoms. For example, C₀ alkyleneindicates a covalent bond and C₁ alkylene is a methylene group. When analkyl residue having a specific number of carbons is named, allgeometric combinations having that number of carbons are intended to beencompassed; thus, for example, “butyl” is meant to include n-butyl,sec-butyl, isobutyl and t-butyl; “propyl” includes n-propyl andisopropyl. “Lower alkyl” refers to alkyl groups having one to fourcarbons.

“Alkenyl” refers to an unsaturated branched or straight-chain alkylgroup having at least one carbon-carbon double bond derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkene. The group may be in either the cis or trans configuration aboutthe double bond(s). Typical alkenyl groups include, but are not limitedto, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl(allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl,2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl; and the like. In certainembodiments, an alkenyl group has from 2 to 20 carbon atoms and in otherembodiments, from 2 to 6 carbon atoms.

“Alkynyl” refers to an unsaturated branched or straight-chain alkylgroup having at least one carbon-carbon triple bond derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkyne. Typical alkynyl groups include, but are not limited to, ethynyl;propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyls such asbut-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl; and the like. In certainembodiments, an alkynyl group has from 2 to 20 carbon atoms and in otherembodiments, from 3 to 6 carbon atoms.

“Cycloalkyl” indicates a non-aromatic carbocyclic ring, usually havingfrom 3 to 7 ring carbon atoms. The ring may be saturated or have one ormore carbon-carbon double bonds. Examples of cycloalkyl groups includecyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, andcyclohexenyl, as well as bridged and caged saturated ring groups such asnorbornane.

By “alkoxy” is meant an alkyl group of the indicated number of carbonatoms attached through an oxygen bridge such as, for example, methoxy,ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy,pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy,2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, and the like. Alkoxy groupswill usually have from 1 to 7 carbon atoms attached through the oxygenbridge. “Lower alkoxy” refers to alkoxy groups having one to fourcarbons.

“Mono- and di-alkylcarboxamide” encompasses a group of the formula—(C═O)NR_(a)R_(b) where R_(a) and R_(b) are independently chosen fromhydrogen and alkyl groups of the indicated number of carbon atoms,provided that R_(a) and R_(b) are not both hydrogen.

“Acyl” refers to the groups (alkyl)-C(O)—; (cycloalkyl)-C(O)—;(aryl)-C(O)—; (heteroaryl)-C(O)—; and (heterocycloalkyl)-C(O)—, whereinthe group is attached to the parent structure through the carbonylfunctionality and wherein alkyl, cycloalkyl, aryl, heteroaryl, andheterocycloalkyl are as described herein. Acyl groups have the indicatednumber of carbon atoms, with the carbon of the keto group being includedin the numbered carbon atoms. For example a C₂ acyl group is an acetylgroup having the formula CH₃(C═O)—.

By “alkoxycarbonyl” is meant a group of the formula (alkoxy)(C═O)—attached through the carbonyl carbon wherein the alkoxy group has theindicated number of carbon atoms. Thus a C₁-C₆ alkoxycarbonyl group isan alkoxy group having from 1 to 6 carbon atoms attached through itsoxygen to a carbonyl linker.

By “amino” is meant the group —NH₂.

“Mono- and di-(alkyl)amino” encompasses secondary and tertiary alkylamino groups, wherein the alkyl groups are as defined above and have theindicated number of carbon atoms. The point of attachment of thealkylamino group is on the nitrogen. Examples of mono- and di-alkylaminogroups include ethylamino, dimethylamino, and methyl-propyl-amino.

The term “aminocarbonyl” refers to the group —CONR^(b)R^(c), where

R^(b) is chosen from H, optionally substituted C₁-C₆ alkyl, optionallysubstituted cycloalkyl, optionally substituted heterocycloalkyl,optionally substituted aryl, and optionally substituted heteroaryl; and

R^(c) is independently chosen from hydrogen and optionally substitutedC₁-C₄ alkyl; or

R^(b) and R^(c) taken together with the nitrogen to which they arebound, form an optionally substituted 5- to 7-memberednitrogen-containing heterocycloalkyl which optionally includes 1 or 2additional heteroatoms selected from O, N, and S in the heterocycloalkylring;

where each substituted group is independently substituted with one ormore substituents independently selected from C₁-C₄ alkyl, aryl,heteroaryl, aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl,—OC₁-C₄ alkyl, —OC₁-C₄ alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁-C₄ haloalkyl,halo, —OH, —NH₂, —C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl),—NH(C₁-C₄ alkyl), —N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄alkylphenyl), cyano, nitro, oxo (as a substitutent for cycloalkyl,heterocycloalkyl, or heteroaryl), —CO₂H, —C(O)OC₁-C₄ alkyl, —CON(C₁-C₄alkyl)(C₁-C₄ alkyl), —CONH(C₁-C₄ alkyl), —CONH₂, —NHC(O)(C₁-C₄ alkyl),—NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), —N(C₁-C₄alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl, —C(O)C₁-C₄ alkylphenyl, —C(O)C₁-C₄haloalkyl, —OC(O)C₁-C₄ alkyl, —SO₂(C₁-C₄ alkyl), —SO₂(phenyl),—SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl), —SO₂NH(phenyl),—NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄ haloalkyl).

“Aryl” encompasses:

6-membered carbocyclic aromatic rings, for example, benzene;

bicyclic ring systems wherein at least one ring is carbocyclic andaromatic, for example, naphthalene, indane, and tetralin; and

tricyclic ring systems wherein at least one ring is carbocyclic andaromatic, for example, fluorene.

For example, aryl includes 6-membered carbocyclic aromatic rings fusedto a 5- to 7-membered heterocycloalkyl ring containing 1 or moreheteroatoms chosen from N, O, and S. For such fused, bicyclic ringsystems wherein only one of the rings is a carbocyclic aromatic ring,the point of attachment may be at the carbocyclic aromatic ring or theheterocycloalkyl ring. Bivalent radicals formed from substituted benzenederivatives and having the free valences at ring atoms are named assubstituted phenylene radicals. Bivalent radicals derived from univalentpolycyclic hydrocarbon radicals whose names end in “-yl” by removal ofone hydrogen atom from the carbon atom with the free valence are namedby adding “-idene” to the name of the corresponding univalent radical,e.g., a naphthyl group with two points of attachment is termednaphthylidene. Aryl, however, does not encompass or overlap in any waywith heteroaryl, separately defined below. Hence, if one or morecarbocyclic aromatic rings is fused with a heterocycloalkyl aromaticring, the resulting ring system is heteroaryl, not aryl, as definedherein.

The term “aryloxy” refers to the group —O-aryl.

“Carbamimidoyl” refers to the group —C(═NH)—NH₂.

“Substituted carbamimidoyl” refers to the group —C(═NR^(e))—NR^(f)R^(g)where R^(e), is chosen from: hydrogen, cyano, optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,optionally substituted heteroaryl, and optionally substitutedheterocycloalkyl; and R^(f) and R^(g) are independently chosen from:hydrogen optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, and optionally substituted heterocycloalkyl, provided thatat least one of R^(e), R^(f), and R^(g) is not hydrogen and whereinsubstituted alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroarylrefer respectively to alkyl, cycloalkyl, aryl, heterocycloalkyl, andheteroaryl wherein one or more (such as up to 5, for example, up to 3)hydrogen atoms are replaced by a substituent independently chosen from:

—R^(a), —OR^(b), optionally substituted amino (including —NR^(c)COR^(b),—NR^(c)CO₂R^(a), —NR^(c)CONR^(b)R^(c), —NR^(b)C(NR^(c))NR^(b)R^(c),—NR^(b)C(NCN)NR^(b)R^(c), and —NR^(c)SO₂R^(a)), halo, cyano, nitro, oxo(as a substitutent for cycloalkyl, heterocycloalkyl, and heteroaryl),optionally substituted acyl (such as —COR^(b)), optionally substitutedalkoxycarbonyl (such as —CO₂R^(b)), aminocarbonyl (such as—CONR^(b)R^(c)), —OCOR^(b), —OCO₂R^(a), —OCONR^(b)R^(c), sulfanyl (suchas SR^(b)), sulfinyl (such as —SOR^(a)), and sulfonyl (such as —SO₂R^(a)and —SO₂NR^(b)R^(c)),

where R^(a) is chosen from optionally substituted C₁-C₆ alkyl,optionally substituted aryl, and optionally substituted heteroaryl;

R^(b) is chosen from H, optionally substituted C₁-C₆ alkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; and

R^(c) is independently chosen from hydrogen and optionally substitutedC₁-C₄ alkyl; or R^(b) and R^(c), and the nitrogen to which they areattached, form an optionally substituted heterocycloalkyl group; and

where each optionally substituted group is unsubstituted orindependently substituted with one or more, such as one, two, or three,substituents independently selected from C₁-C₄ alkyl, aryl, heteroaryl,aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, —OC₁-C₄alkyl, —OC₁-C₄ alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁-C₄ haloalkyl, halo,—OH, —NH₂, —C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄alkyl), —N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄ alkylphenyl),cyano, nitro, oxo (as a substitutent for cycloalkyl, heterocycloalkyl,or heteroaryl), —CO₂H, —C(O)OC₁-C₄ alkyl, —CON(C₁-C₄ alkyl)(C₁-C₄alkyl), —CONH(C₁-C₄ alkyl), —CONH₂, —NHC(O)(C₁-C₄ alkyl),—NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), —N(C₁-C₄alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl, —C(O)C₁-C₄ phenyl, —C(O)C₁-C₄haloalkyl, —OC(O)C₁-C₄ alkyl, —SO₂(C₁-C₄ alkyl), —SO₂(phenyl),—SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl), —SO₂NH(phenyl),—NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄ haloalkyl).

The term “halo” includes fluoro, chloro, bromo, and iodo, and the term“halogen” includes fluorine, chlorine, bromine, and iodine.

“Haloalkyl” indicates alkyl as defined above having the specified numberof carbon atoms, substituted with 1 or more halogen atoms, up to themaximum allowable number of halogen atoms. Examples of haloalkylinclude, but are not limited to, trifluoromethyl, difluoromethyl,2-fluoroethyl, and penta-fluoroethyl.

“Heteroaryl” encompasses:

5- to 7-membered aromatic, monocyclic rings containing one or more, forexample, from 1 to 4, or in certain embodiments, from 1 to 3,heteroatoms chosen from N, O, and S, with the remaining ring atoms beingcarbon;

bicyclic heterocycloalkyl rings containing one or more, for example,from 1 to 4, or in certain embodiments, from 1 to 3, heteroatoms chosenfrom N, O, and S, with the remaining ring atoms being carbon and whereinat least one heteroatom is present in an aromatic ring; and

tricyclic heterocycloalkyl rings containing one or more, for example,from 1 to 5, or in certain embodiments, from 1 to 4, heteroatoms chosenfrom N, O, and S, with the remaining ring atoms being carbon and whereinat least one heteroatom is present in an aromatic ring.

For example, heteroaryl includes a 5- to 7-membered heterocycloalkyl,aromatic ring fused to a 5- to 7-membered cycloalkyl or heterocycloalkylring. For such fused, bicyclic heteroaryl ring systems wherein only oneof the rings contains one or more heteroatoms, the point of attachmentmay be at either ring. When the total number of S and O atoms in theheteroaryl group exceeds 1, those heteroatoms are not adjacent to oneanother. In certain embodiments, the total number of S and O atoms inthe heteroaryl group is not more than 2. In certain embodiments, thetotal number of S and O atoms in the aromatic heterocycle is not morethan 1. Examples of heteroaryl groups include, but are not limited to,(as numbered from the linkage position assigned priority 1), 2-pyridyl,3-pyridyl, 4-pyridyl, 2,3-pyrazinyl, 3,4-pyrazinyl, 2,4-pyrimidinyl,3,5-pyrimidinyl, 2,3-pyrazolinyl, 2,4-imidazolinyl, isoxazolinyl,oxazolinyl, thiazolinyl, thiadiazolinyl, tetrazolyl, thienyl,benzothiophenyl, furanyl, benzofuranyl, benzoimidazolinyl, indolinyl,pyridazinyl, triazolyl, quinolinyl, pyrazolyl, and5,6,7,8-tetrahydroisoquinolinyl. Bivalent radicals derived fromunivalent heteroaryl radicals whose names end in “-yl” by removal of onehydrogen atom from the atom with the free valence are named by adding“-idene” to the name of the corresponding univalent radical, e.g., apyridyl group with two points of attachment is a pyridylidene.Heteroaryl does not encompass or overlap with aryl, cycloalkyl, orheterocycloalkyl, as defined herein

Substituted heteroaryl also includes ring systems substituted with oneor more oxide (—O⁻) substituents, such as pyridinyl N-oxides.

By “heterocycloalkyl” is meant a single, non-aromatic ring, usually with3 to 7 ring atoms, containing at least 2 carbon atoms in addition to 1-3heteroatoms independently selected from oxygen, sulfur, and nitrogen, aswell as combinations comprising at least one of the foregoingheteroatoms. The ring may be saturated or have one or more carbon-carbondouble bonds. Suitable heterocycloalkyl groups include, for example (asnumbered from the linkage position assigned priority 1), 2-pyrrolidinyl,2,4-imidazolidinyl, 2,3-pyrazolidinyl, 2-piperidyl, 3-piperidyl,4-piperidyl, and 2,5-piperizinyl. Morpholinyl groups are alsocontemplated, including 2-morpholinyl and 3-morpholinyl (numberedwherein the oxygen is assigned priority 1). Substituted heterocycloalkylalso includes ring systems substituted with one or more oxo (═O) oroxide (—O⁻) substituents, such as piperidinyl N-oxide,morpholinyl-N-oxide, 1-oxo-1-thiomorpholinyl and1,1-dioxo-1-thiomorpholinyl.

“Heterocycloalkyl” also includes bicyclic ring systems wherein onenon-aromatic ring, usually with 3 to 7 ring atoms, contains at least 2carbon atoms in addition to 1-3 heteroatoms independently selected fromoxygen, sulfur, and nitrogen, as well as combinations comprising atleast one of the foregoing heteroatoms; and the other ring, usually with3 to 7 ring atoms, optionally contains 1-3 heteroatoms independentlyselected from oxygen, sulfur, and nitrogen and is not aromatic.

As used herein, “modulation” refers to a change in activity as a director indirect response to the presence of a chemical entity as describedherein, relative to the activity of in the absence of the chemicalentity. The change may be an increase in activity or a decrease inactivity, and may be due to the direct interaction of the chemicalentity with the a target or due to the interaction of the chemicalentity with one or more other factors that in turn affect the target'sactivity. For example, the presence of the chemical entity may, forexample, increase or decrease the target activity by directly binding tothe target, by causing (directly or indirectly) another factor toincrease or decrease the target activity, or by (directly or indirectly)increasing or decreasing the amount of target present in the cell ororganism.

The term “sulfanyl” includes the groups: —S-(optionally substituted(C₁-C₆)alkyl), —S-(optionally substituted aryl), —S-(optionallysubstituted heteroaryl), and —S-(optionally substitutedheterocycloalkyl). Hence, sulfanyl includes the group C₁-C₆alkylsulfanyl.

The term “sulfinyl” includes the groups: —S(O)-(optionally substituted(C₁-C₆)alkyl), —S(O)-optionally substituted aryl), —S(O)-optionallysubstituted heteroaryl), —S(O)-(optionally substitutedheterocycloalkyl); and —S(O)-(optionally substituted amino).

The term “sulfonyl” includes the groups: —S(O₂)-(optionally substituted(C₁-C₆)alkyl), —S(O₂)-optionally substituted aryl), —S(O₂)-optionallysubstituted heteroaryl), —S(O₂)-(optionally substitutedheterocycloalkyl) and —S(O₂)-(optionally substituted amino).

The term “substituted”, as used herein, means that any one or morehydrogens on the designated atom or group is replaced with a selectionfrom the indicated group, provided that the designated atom's normalvalence is not exceeded. When a substituent is oxo (i.e., ═O) then 2hydrogens on the atom are replaced. Combinations of substituents and/orvariables are permissible only if such combinations result in stablecompounds or useful synthetic intermediates. A stable compound or stablestructure is meant to imply a compound that is sufficiently robust tosurvive isolation from a reaction mixture, and subsequent formulation asan agent having at least practical utility. Unless otherwise specified,substituents are named into the core structure. For example, it is to beunderstood that when (cycloalkyl)alkyl is listed as a possiblesubstituent, the point of attachment of this substituent to the corestructure is in the alkyl portion.

The terms “substituted” alkyl, cycloalkyl, aryl, heterocycloalkyl, andheteroaryl, unless otherwise expressly defined, refer respectively toalkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl wherein one ormore (such as up to 5, for example, up to 3) hydrogen atoms are replacedby a substituent independently chosen from:

—R^(a), —OR^(b), optionally substituted amino (including —NR^(c)COR^(b),—NR^(c)CO₂R^(a), —NR^(c)CONR^(b)R^(c), —NR^(b)C(NR^(c))NR^(b)R^(c),—NR^(b)C(NCN)NR^(b)R^(c), and —NR^(c)SO₂R^(a)), halo, cyano, nitro, oxo(as a substitutent for cycloalkyl, heterocycloalkyl, and heteroaryl),optionally substituted acyl (such as —COR^(b)), optionally substitutedalkoxycarbonyl (such as —CO₂R^(b)), aminocarbonyl (such as—CONR^(b)R^(c)), —OCOR^(b), —OCO₂R^(a), —OCONR^(b)R^(c), sulfanyl (suchas SR^(b)), sulfinyl (such as —SOR^(a)), and sulfonyl (such as —SO₂R^(a)and —SO₂NR^(b)R^(c)),

where R^(a) is chosen from optionally substituted C₁-C₆ alkyl,optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted alkenyl, optionally substitutedalkynyl, optionally substituted aryl, and optionally substitutedheteroaryl;

R^(b) is chosen from hydrogen, optionally substituted C₁-C₆ alkyl,optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; and

R^(c) is independently chosen from hydrogen and optionally substitutedC₁-C₄ alkyl; or

R^(b) and R^(c), and the nitrogen to which they are attached, form anoptionally substituted heterocycloalkyl group; and

where each optionally substituted group is unsubstituted orindependently substituted with one or more, such as one, two, or three,substituents independently selected from C₁-C₄ alkyl, aryl, heteroaryl,aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, —OC₁-C₄alkyl, —OC₁-C₄ alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁-C₄ haloalkyl, halo,—OH, —NH₂, —C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄alkyl), —N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄ alkylphenyl),cyano, nitro, oxo (as a substitutent for cycloalkyl, heterocycloalkyl,or heteroaryl), —CO₂H, —C(O)OC₁-C₄ alkyl, —CON(C₁-C₄ alkyl)(C₁-C₄alkyl), —CONH(C₁-C₄ alkyl), —CONH₂, —NHC(O)(C₁-C₄ alkyl),—NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), —N(C₁-C₄alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl, —C(O)C₁-C₄ alkylphenyl, —C(O)C₁-C₄haloalkyl, —OC(O)C₁-C₄ alkyl, —SO₂(C₁-C₄ alkyl), —SO₂(phenyl),—SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl), —SO₂NH(phenyl),—NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄ haloalkyl).

The term “substituted acyl” refers to the groups (substitutedalkyl)-C(O)—; (substituted cycloalkyl)-C(O)—; (substituted aryl)-C(O)—;(substituted heteroaryl)-C(O)—; and (substitutedheterocycloalkyl)-C(O)—, wherein the group is attached to the parentstructure through the carbonyl functionality and wherein substitutedalkyl, cycloalkyl, aryl, heteroaryl, and heterocycloalkyl, referrespectively to alkyl, cycloalkyl, aryl, heteroaryl, andheterocycloalkyl wherein one or more (such as up to 5, for example, upto 3) hydrogen atoms are replaced by a substituent independently chosenfrom:

—R^(a), —OR^(b), optionally substituted amino (including —NR^(c)COR^(b),—NR^(c)CO₂R^(a), —NR^(c)CONR^(b)R^(c), —NR^(b)C(NR^(c))NR^(b)R^(c),—NR^(b)C(NCN)NR^(b)R^(c), and —NR^(c)SO₂R^(a)), halo, cyano, nitro, oxo(as a substitutent for cycloalkyl, heterocycloalkyl, and heteroaryl),optionally substituted acyl (such as —COR^(b)), optionally substitutedalkoxycarbonyl (such as —CO₂R^(b)), aminocarbonyl (such as—CONR^(b)R^(c)), —OCOR^(b), —OCO₂R^(a), —OCONR^(b)R^(c), sulfanyl (suchas SR^(b)), sulfinyl (such as —SOR^(a)), and sulfonyl (such as —SO₂R^(a)and —SO₂NR^(b)R^(c)),

where R^(a) is chosen from optionally substituted C₁-C₆ alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, and optionally substituted heteroaryl;

R^(b) is chosen from H, optionally substituted C₁-C₆ alkyl, optionallysubstituted cycloalkyl, optionally substituted heterocycloalkyl,optionally substituted aryl, and optionally substituted heteroaryl; and

R^(c) is independently chosen from hydrogen and optionally substitutedC₁-C₄ alkyl; or

R^(b) and R^(c), and the nitrogen to which they are attached, form anoptionally substituted heterocycloalkyl group; and

where each optionally substituted group is unsubstituted orindependently substituted with one or more, such as one, two, or three,substituents independently selected from C₁-C₄ alkyl, aryl, heteroaryl,aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, —OC₁-C₄alkyl, —OC₁-C₄ alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁-C₄ haloalkyl, halo,—OH, —NH₂, —C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄alkyl), —N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄ alkylphenyl),cyano, nitro, oxo (as a substitutent for cycloalkyl, heterocycloalkyl,or heteroaryl), —CO₂H, —C(O)OC₁-C₄ alkyl, —CON(C₁-C₄ alkyl)(C₁-C₄alkyl), —CONH(C₁-C₄ alkyl), —CONH₂, —NHC(O)(C₁-C₄ alkyl),—NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), —N(C₁-C₄alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl, —C(O)C₁-C₄ alkylphenyl, —C(O)C₁-C₄haloalkyl, —OC(O)C₁-C₄ alkyl, —SO₂(C₁-C₄ alkyl), —SO₂(phenyl),—SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl), —SO₂NH(phenyl),—NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄ haloalkyl).

The term “substituted alkoxy” refers to alkoxy wherein the alkylconstituent is substituted (i.e., —O-(substituted alkyl)) wherein“substituted alkyl” refers to alkyl wherein one or more (such as up to5, for example, up to 3) hydrogen atoms are replaced by a substituentindependently chosen from:

—R^(a), —OR^(b), optionally substituted amino (including —NR^(c)COR^(b),—NR^(c)CO₂R^(a), —NR^(c)CONR^(b)R^(c), —NR^(b)C(NR^(c))NR^(b)R^(c),—NR^(b)C(NCN)NR^(b)R^(c), and —NR^(c)SO₂R^(a)), halo, cyano, nitro, oxo(as a substitutent for cycloalkyl, heterocycloalkyl, and heteroaryl),optionally substituted acyl (such as —COR^(b)), optionally substitutedalkoxycarbonyl (such as —CO₂R^(b)), aminocarbonyl (such as—CONR^(b)R^(c)), —OCOR^(b), —OCO₂R^(a), —OCONR^(b)R^(c), sulfanyl (suchas SR^(b)), sulfinyl (such as —SOR^(a)), and sulfonyl (such as —SO₂R^(a)and —SO₂NR^(b)R^(c)),

where R^(a) is chosen from optionally substituted C₁-C₆ alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, and optionally substituted heteroaryl;

R^(b) is chosen from H, optionally substituted C₁-C₆ alkyl, optionallysubstituted cycloalkyl, optionally substituted heterocycloalkyl,optionally substituted aryl, and optionally substituted heteroaryl; and

R^(c) is independently chosen from hydrogen and optionally substitutedC₁-C₄ alkyl; or

R^(b) and R^(c), and the nitrogen to which they are attached, form anoptionally substituted heterocycloalkyl group; and

where each optionally substituted group is unsubstituted orindependently substituted with one or more, such as one, two, or three,substituents independently selected from C₁-C₄ alkyl, aryl, heteroaryl,aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, —OC₁-C₄alkyl, —OC₁-C₄ alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁-C₄ haloalkyl, halo,—OH, —NH₂, —C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄alkyl), —N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄ alkylphenyl),cyano, nitro, oxo (as a substitutent for cycloalkyl, heterocycloalkyl,or heteroaryl), —CO₂H, —C(O)OC₁-C₄ alkyl, —CON(C₁-C₄ alkyl)(C₁-C₄alkyl), —CONH(C₁-C₄ alkyl), —CONH₂, —NHC(O)(C₁-C₄ alkyl),—NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), —N(C₁-C₄alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl, —C(O)C₁-C₄ alkylphenyl, —C(O)C₁-C₄haloalkyl, —OC(O)C₁-C₄ alkyl, —SO₂(C₁-C₄ alkyl), —SO₂(phenyl),—SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl), —SO₂NH(phenyl),—NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄ haloalkyl). Insome embodiments, a substituted alkoxy group is “polyalkoxy” or—O-(optionally substituted alkylene)-(optionally substituted alkoxy),and includes groups such as —OCH₂CH₂OCH₃, and residues of glycol etherssuch as polyethyleneglycol, and —O(CH₂CH₂O)_(x)CH₃, where x is aninteger of 2-20, such as 2-10, and for example, 2-5. Another substitutedalkoxy group is hydroxyalkoxy or —OCH₂(CH₂)_(y)OH, where y is an integerof 1-10, such as 1-4.

The term “substituted alkoxycarbonyl” refers to the group (substitutedalkyl)-O—C(O)— wherein the group is attached to the parent structurethrough the carbonyl functionality and wherein substituted refers toalkyl wherein one or more (such as up to 5, for example, up to 3)hydrogen atoms are replaced by a substituent independently chosen from:

—R^(a), —OR^(b), optionally substituted amino (including —NR^(c)COR^(b),—NR^(c)CO₂R^(a), —NR^(c)CONR^(b)R^(c), —NR^(b)C(NR^(c))NR^(b)R^(c),—NR^(b)C(NCN)NR^(b)R^(c), and —NR^(c)SO₂R^(a)), halo, cyano, nitro, oxo(as a substitutent for cycloalkyl, heterocycloalkyl, and heteroaryl),optionally substituted acyl (such as —COR^(b)), optionally substitutedalkoxycarbonyl (such as —CO₂R^(b)), aminocarbonyl (such as—CONR^(b)R^(c)), —OCOR^(b), —OCO₂R^(a), —OCONR^(b)R^(c), sulfanyl (suchas SR^(b)), sulfinyl (such as —SOR^(a)), and sulfonyl (such as —SO₂R^(a)and —SO₂NR^(b)R^(c)),

where R^(a) is chosen from optionally substituted C₁-C₆ alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, and optionally substituted heteroaryl;

R^(b) is chosen from H, optionally substituted C₁-C₆ alkyl, optionallysubstituted cycloalkyl, optionally substituted heterocycloalkyl,optionally substituted aryl, and optionally substituted heteroaryl; and

R^(c) is independently chosen from hydrogen and optionally substitutedC₁-C₄ alkyl; or

R^(b) and R^(c), and the nitrogen to which they are attached, form anoptionally substituted heterocycloalkyl group; and

where each optionally substituted group is unsubstituted orindependently substituted with one or more, such as one, two, or three,substituents independently selected from C₁-C₄ alkyl, aryl, heteroaryl,aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, —OC₁-C₄alkyl, —OC₁-C₄ alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁-C₄ haloalkyl, halo,—OH, —NH₂, —C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄alkyl), —N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄ alkylphenyl),cyano, nitro, oxo (as a substitutent for cycloalkyl, heterocycloalkyl,or heteroaryl), —CO₂H, —C(O)OC₁-C₄ alkyl, —CON(C₁-C₄ alkyl)(C₁-C₄alkyl), —CONH(C₁-C₄ alkyl), —CONH₂, —NHC(O)(C₁-C₄ alkyl),—NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), —N(C₁-C₄alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl, —C(O)C₁-C₄ alkylphenyl, —C(O)C₁-C₄haloalkyl, —OC(O)C₁-C₄ alkyl, —SO₂(C₁-C₄ alkyl), —SO₂(phenyl),—SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl), —SO₂NH(phenyl),—NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄ haloalkyl).

The term “substituted amino” refers to the group —NHR^(d) or—NR^(d)R^(e) wherein R^(d) is chosen from: hydroxy, optionallysubstituted alkoxy, optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted acyl, optionally substitutedcarbamimidoyl, optionally substituted aminocarbonyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted heterocycloalkyl, optionally substituted alkoxycarbonyl,optionally substituted sulfinyl and optionally substituted sulfonyl, andwherein R^(e) is chosen from: optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aminocarbonyl, optionallysubstituted acyl, optionally substituted aryl, optionally substitutedheteroaryl, and optionally substituted heterocycloalkyl, and whereinsubstituted alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroarylrefer respectively to alkyl, cycloalkyl, aryl, heterocycloalkyl, andheteroaryl wherein one or more (such as up to 5, for example, up to 3)hydrogen atoms are replaced by a substituent independently chosen from:

—R^(a), —OR^(b), optionally substituted amino (including —NR^(c)COR^(b),—NR^(c)CO₂R^(a), —NR^(c)CONR^(b)R^(c), —NR^(b)C(NR^(c))NR^(b)R^(c),—NR^(b)C(NCN)NR^(b)R^(c), and —NR^(c)SO₂R^(a)), halo, cyano, nitro, oxo(as a substitutent for cycloalkyl, heterocycloalkyl, and heteroaryl),optionally substituted acyl (such as —COR^(b)), optionally substitutedalkoxycarbonyl (such as —CO₂R^(b)), aminocarbonyl (such as—CONR^(b)R^(c)), —OCOR^(b), —OCO₂R^(a), —OCONR^(b)R^(c), sulfanyl (suchas SR^(b)), sulfinyl (such as —SOR^(a)), and sulfonyl (such as —SO₂R^(a)and —SO₂NR^(b)R^(c)),

where R^(a) is chosen from optionally substituted C₁-C₆ alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, and optionally substituted heteroaryl;

R^(b) is chosen from H, optionally substituted C₁-C₆ alkyl, optionallysubstituted cycloalkyl, optionally substituted heterocycloalkyl,optionally substituted aryl, and optionally substituted heteroaryl; and

R^(c) is independently chosen from hydrogen and optionally substitutedC₁-C₄ alkyl; or

R^(b) and R^(c), and the nitrogen to which they are attached, form anoptionally substituted heterocycloalkyl group; and

where each optionally substituted group is unsubstituted orindependently substituted with one or more, such as one, two, or three,substituents independently selected from C₁-C₄ alkyl, aryl, heteroaryl,aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, —OC₁-C₄alkyl, —OC₁-C₄ alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁-C₄ haloalkyl, halo,—OH, —NH₂, —C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄alkyl), —N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄ alkylphenyl),cyano, nitro, oxo (as a substitutent for cycloalkyl, heterocycloalkyl,or heteroaryl), —CO₂H, —C(O)OC₁-C₄ alkyl, —CON(C₁-C₄ alkyl)(C₁-C₄alkyl), —CONH(C₁-C₄ alkyl), —CONH₂, —NHC(O)(C₁-C₄ alkyl),—NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), —N(C₁-C₄alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl, —C(O)C₁-C₄ alkylphenyl, —C(O)C₁-C₄haloalkyl, —OC(O)C₁-C₄ alkyl, —SO₂(C₁-C₄ alkyl), —SO₂(phenyl),—SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl), —SO₂NH(phenyl),—NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄ haloalkyl); and

wherein optionally substituted acyl, optionally substitutedalkoxycarbonyl, sulfinyl and sulfonyl are as defined herein.

The term “substituted amino” also refers to N-oxides of the groups—NHR^(d), and NR^(d)R^(d) each as described above. N-oxides can beprepared by treatment of the corresponding amino group with, forexample, hydrogen peroxide or m-chloroperoxybenzoic acid. The personskilled in the art is familiar with reaction conditions for carrying outthe N-oxidation.

Compounds of Formula I include, but are not limited to, optical isomersof compounds of Formula I, racemates, and other mixtures thereof. Inthose situations, the single enantiomers or diastereomers, i.e.,optically active forms, can be obtained by asymmetric synthesis or byresolution of the racemates. Resolution of the racemates can beaccomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent, or chromatography,using, for example a chiral high-pressure liquid chromatography (HPLC)column. In addition, compounds of Formula I include Z- and E-forms (orcis- and trans-forms) of compounds with carbon-carbon double bonds.Where compounds of Formula I exists in various tautomeric forms,chemical entities of the present invention include all tautomeric formsof the compound.

Chemical entities of the present invention include, but are not limitedto compounds of Formula I and all pharmaceutically acceptable formsthereof. Pharmaceutically acceptable forms of the chemical entitiesrecited herein include pharmaceutically acceptable salts, solvates,crystal forms (including polymorphs and clathrates), chelates,non-covalent complexes, prodrugs, and mixtures thereof. In certainembodiments, the compounds described herein are in the form ofpharmaceutically acceptable salts. Hence, the terms “chemical entity”and “chemical entities” also encompass pharmaceutically acceptablesalts, solvates, chelates, non-covalent complexes, prodrugs, andmixtures.

“Pharmaceutically acceptable salts” include, but are not limited tosalts with inorganic acids, such as hydrochloride, phosphate,diphosphate, hydrobromide, sulfate, sulfinate, nitrate, and like salts;as well as salts with an organic acid, such as malate, maleate,fumarate, tartrate, succinate, citrate, lactate, methanesulfonate,p-toluenesulfonate, 2-hydroxyethylsulfonate, benzoate, salicylate,stearate, and alkanoate such as acetate, HOOC—(CH₂)_(n)—COOH where n is0-4, and like salts. Similarly, pharmaceutically acceptable cationsinclude, but are not limited to sodium, potassium, calcium, aluminum,lithium, and ammonium.

In addition, if the compound of Formula I is obtained as an acidaddition salt, the free base can be obtained by basifying a solution ofthe acid salt. Conversely, if the product is a free base, an additionsalt, particularly a pharmaceutically acceptable addition salt, may beproduced by dissolving the free base in a suitable organic solvent andtreating the solution with an acid, in accordance with conventionalprocedures for preparing acid addition salts from base compounds. Thoseskilled in the art will recognize various synthetic methodologies thatmay be used to prepare non-toxic pharmaceutically acceptable additionsalts.

As noted above, prodrugs also fall within the scope of chemicalentities, for example ester or amide derivatives of the compounds ofFormula I. The term “prodrugs” includes any chemical entities thatbecome compounds of Formula I when administered to a patient, e.g., uponmetabolic processing of the prodrug. Examples of prodrugs include, butare not limited to, acetate, formate, phosphate, and benzoate and likederivatives of functional groups (such as alcohol or amine groups) inthe compounds of Formula I.

The term “solvate” refers to the chemical entity formed by theinteraction of a solvent and a compound. Suitable solvates arepharmaceutically acceptable solvates, such as hydrates, includingmonohydrates and hemi-hydrates.

The term “chelate” refers to the chemical entity formed by thecoordination of a compound to a metal ion at two (or more) points.

The term “non-covalent complex” refers to the chemical entity formed bythe interaction of a compound and another molecule wherein a covalentbond is not formed between the compound and the molecule. For example,complexation can occur through van der Waals interactions, hydrogenbonding, and electrostatic interactions (also called ionic bonding).

The term “active agent” is used to indicate a chemical entity which hasbiological activity. In certain embodiments, an “active agent” is acompound having pharmaceutical utility. For example an active agent maybe an anti-cancer therapeutic.

By “significant” is meant any detectable change that is statisticallysignificant in a standard parametric test of statistical significancesuch as Student's T-test, where p<0.05.

The term “therapeutically effective amount” of a chemical entity of thisinvention means an amount effective, when administered to a human ornon-human patient, to provide a therapeutic benefit such as ameliorationof symptoms, slowing of disease progression, or prevention of disease.

“Treatment” or “treating” means any treatment of a disease in a patient,including:

-   -   a) preventing the disease, that is, causing the clinical        symptoms of the disease not to develop;    -   b) inhibiting the disease;    -   c) slowing or arresting the development of clinical symptoms;        and/or    -   d) relieving the disease, that is, causing the regression of        clinical symptoms.

“Patient” refers to an animal, such as a mammal, that has been or willbe the object of treatment, observation or experiment. The methods ofthe invention can be useful in both human therapy and veterinaryapplications. In some embodiments, the patient is a mammal; in someembodiments the patient is human; and in some embodiments the patient ischosen from cats and dogs.

The present invention is directed to at least one chemical entity thatis a selective modulator of the cardiac sarcomere (e.g., by stimulatingor otherwise potentiating the activity of cardiac myosin).

Compounds having the structure of Formula I can be named and numbered(e.g., using ChemInnovation's Pipeline Pilot in connection with Chem 4-DDraw and the Nomenclator Module).

The present invention provides at least one chemical entity chosen fromcompounds of Formula I:

and pharmaceutically acceptable salts, solvates, chelates, non-covalentcomplexes, prodrugs, and mixtures thereof,

where one of Z¹ and Z² is —NR¹⁵C(O)NR¹⁶R⁴ and the other of Z¹ and Z² isR³;

R⁴ is chosen from optionally substituted aryl, optionally substitutedaralkyl; optionally substituted cycloalkyl, optionally substitutedheteroaryl, optionally substituted heteroaralkyl and optionallysubstituted heterocycloalkyl;

R³ is chosen from hydrogen, halo, cyano, hydroxyl, optionallysubstituted alkyl, optionally substituted heterocycloalkyl, andoptionally substituted heteroaryl;

R¹ and R² are independently chosen from hydrogen, halo, cyano,optionally substituted alkyl, optionally substituted heterocycloalkyl,and optionally substituted heteroaryl;

R¹⁵ and R¹⁶ are independently chosen from hydrogen, and optionallysubstituted alkyl;

W¹ is chosen from N and C;

A is chosen from cycloalkyl, heterocycloalkyl, aryl, and heteroarylgroups having from 5 to 7 ring atoms including the atoms shared with the6 membered aromatic ring containing W¹;

R⁵ is chosen from optionally substituted alkyl, optionally substitutedamino, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted cycloalkyl and optionally substitutedheterocycloalkyl;

p is 0 or 1;

R⁶ is chosen from optionally substituted alkyl and halo;

L is chosen from a bond, optionally substituted lower alkylene, —O—,—O-(optionally substituted lower alkylene)-, -(optionally substitutedlower alkylene)-O—, —S—, —S-(optionally substituted lower alkylene)-,-(optionally substituted lower alkylene)-S—, —SO₂—, —SO₂-(optionallysubstituted lower alkylene)-, and -(optionally substituted loweralkylene)-SO₂—

provided that if R⁵ is amino or if R⁵ is heteroaryl or heterocycloalkylwith a heteroatom bonded to L, then L is not —O—, —S—, —O-alkyl, or—S-alkyl.

In certain embodiments, Z¹ is —CHNHC(O)NHR⁴ and Z² is R³.

In certain embodiments, Z² is —CHNHC(O)NHR⁴ and Z¹ is R³.

In certain embodiments, R⁴ is chosen from optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted heteroaryl andoptionally substituted heterocycloalkyl.

In some embodiments, R⁴ is chosen from optionally substituted phenyl,optionally substituted naphthyl, optionally substituted pyrrolyl,optionally substituted thiazolyl, optionally substituted isoxazolyl,optionally substituted pyrazolyl, optionally substituted oxazolyl,optionally substituted 1,3,4-oxadiazolyl, optionally substitutedpyridinyl, optionally substituted pyrazinyl, optionally substitutedpyrimidinyl and optionally substituted pyridazinyl.

In some embodiments, R⁴ is chosen from optionally substituted pyridinyl.

In some embodiments, R⁴ is chosen from 6-methoxy-pyridin-3-yl,6-methyl-pyridin-3-yl and pyridin-3-yl.

In some embodiments, R³ is chosen from hydrogen, halo, cyano, loweralkyl, and hydroxyl.

In some embodiments, R³ is chosen from hydrogen, fluoro, chloro, methyl,ethyl and hydroxyl.

In some embodiments, R³ is hydrogen.

In some embodiments, R¹ and R² are independently chosen from halo, cyanoand lower alkyl.

In some embodiments, R² is hydrogen.

In some embodiments, R³ is hydrogen.

In some embodiments, R¹, R² and R³ are hydrogen.

In some embodiments, W¹ is N. In some embodiments, W¹ is C.

In some embodiments, A is a five-membered cycloalkyl, five-memberedheteroaryl, and five-membered heterocycloalkyl rings.

In some embodiments, A is a five membered ring selected from

where the bonds with dashed lines across them represent the connectivityto 6 membered aromatic ring.

In some embodiments, R⁵ is selected from optionally substitutedpiperazinyl; optionally substituted1,1-dioxo-1λ⁶-[1,2,5]thiadiazolidin-2-yl; optionally substituted3-oxo-tetrahydro-pyrrolo[1,2-c]oxazol-6-yl, optionally substituted2-oxo-imidazolidin-1-yl; optionally substituted morpholinyl; optionallysubstituted 1,1-dioxo-1λ⁶-thiomorpholin-4-yl; optionally substitutedpyrrolidinyl; optionally substituted piperidinyl; optionally substitutedazepanyl; optionally substituted 1,4-diazepanyl; optionally substituted3-oxo-tetrahydro-1H-oxazolo[3,4-a]pyrazin-3(5H)-one; optionallysubstituted 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, andoptionally substituted

wherein R^(A) and R^(B) are independently hydrogen, optionallysubstituted alkyl, or R^(A) and R^(B) taken together with the carbon towhich they are attached, form an optionally substituted 3- to 7-memberedring which optionally incorporates one or two additional heteroatoms,selected from N, O, and S in the ring.

In some embodiments, R⁵ is optionally substituted piperazinyl.

In some embodiments, R⁵ is chosen from4-(dimethylcarbamoyl)piperazine-1-yl,4-(N,N-dimethylsulfamoyl)piperazine-1-yl, 4-acetyl-piperazin-1-yl,4-ethoxycarbonyl-piperazin-1-yl, 4-ethylsulfonyl-piperazin-1-yl,4-methoxycarbonyl-piperazin-1-yl, 4-methylsulfonyl-piperazin-1-yl,4-t-butoxycarbonyl-piperazin-1-yl, piperazin-1-yl,4-(4-acetylpiperazine-1-carbonyl)piperazin-1-yl,4-(4-methylpiperazine-1-carbonyl)piperazin-1-yl,4-(piperidine-1-carbonyl)piperazin-1-yl,4-(morpholine-4-carbonyl)piperazin-1-yl,4-(cyclobutylsulfonyl)-piperazin-1-yl, 4-(ethylsulfonyl)piperazin-1-yl,4-(isopropylsulfonyl)piperazin-1-yl,4-(cyclopropylsulfonyl)piperazin-1-yl, and 4-(1,1-dioxidethiomorpholine-4-carbonyl)piperazin-1-yl.

In some embodiments, R⁵ is optionally substituted amino.

In some embodiments, R⁵ is selected from optionally substituted amino ofthe Formula NR⁹R¹⁰ where R⁹ is selected from hydrogen, optionallysubstituted alkyl, optionally substituted acyl, optionally substitutedalkoxycarbonyl, optionally substituted aminocarbonyl, and optionallysubstituted sulfonyl, and R¹⁰ is selected from hydrogen and optionallysubstituted alkyl.

In some embodiments, R⁹ is —(SO₂)—R¹⁷ wherein R¹⁷ is lower alkyl or—NR¹¹R¹² wherein R¹¹ and R¹² are independently hydrogen or lower alkyl.

In some embodiments, R⁹ is optionally substituted lower alkoxycarbonyl.

In some embodiments, R⁹ is lower alkyl.

In some embodiments, R⁹ is acetyl.

In some embodiments, R¹⁰ is selected from hydrogen, methyl, ethyl andmethoxycarbonyl.

In some embodiments, R⁵ is selected from amino, methylamino,2-(methoxycarbonylamino), 2-(tert-butoxycarbonylamino),benzyloxycarbonylamino, ethylsulfonamido, N,N-dimethylsulfamoylamino,acetylamino, 3,3-dimethylureido, methoxycarbonyl(methyl)amino,N,N-diethylamino, N-methylethylsulfonamido, N-acetyl-N-methylamino,N-t-butoxycarbonyl-N-methylamino, (N,N-dimethylsulfamoyl)(methyl)amino,1,3,3-trimethylureido, and bis(methoxycarbonyl)amino.

In some embodiments, p is 0.

In some embodiments, p is 1.

In some embodiments, R⁶ is chosen from lower alkyl and halo.

In some embodiments, L is chosen from a bond, and optionally substitutedalkylene.

In some embodiments, L is a bond.

In some embodiments, L is —CH₂—.

In some embodiments, L is —CH₂CH₂—.

In some embodiments, L is —CH₂CH₂CH₂—.

In some embodiments, the combinations of p R⁶, R⁵, L and ring A areselected from

where the bonds with dashed lines across them represent the connectivityto 6 membered aromatic ring.

In some embodiments, A is selected from six-membered cycloalkyl,six-membered aryl, six-membered heterocycloalkyl, and six-memberedheteroaryl rings.

Also provided is at least one chemical entity chosen from compounds ofFormula Ic

and pharmaceutically acceptable salts, solvates, chelates, non-covalentcomplexes, prodrugs, and mixtures thereof, where p, L, W¹, R¹, R², R³,R⁴, R⁵, R⁶, R¹⁵ and R¹⁶ are as described for compounds of Formula I andW² is C or NH.

In some embodiments, W² is C.

In some embodiments, W² is NH.

In some embodiments, W¹ and W² are C, p is 0 and R⁶ is absent.

In some embodiments, p is 0, R⁶ is absent, R⁵-L is bonded to W², W¹ isC, and W² is N.

Also provided is at least one chemical entity is chosen from compoundsof Formula Ia

and pharmaceutically acceptable salts, solvates, chelates, non-covalentcomplexes, prodrugs, and mixtures thereof, where A p, L, W¹, R¹, R², R³,R⁴, R⁵ and R⁶ are as described for compounds of Formula I.

Also provided is at least one chemical entity is chosen from compoundsof Formula Ib.

and pharmaceutically acceptable salts, solvates, chelates, non-covalentcomplexes, prodrugs, and mixtures thereof, where A p, L, W¹, R¹, R², R³,R⁴, R⁵, R⁶, R¹⁵ and R¹⁶ are as described for compounds of Formula I.

In some embodiments, the compound of Formula I is chosen from compoundsin Tables I and II below.

TABLE I

R⁶ R⁵ L A W¹ R¹ R² R³ R⁴ H 4- methoxy- carbonyl- piperazin- 1-yl bond

C H H H 6-methyl- pyridin-3- yl H 4-t- butoxy- carbonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H 4- methoxy- carbonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H 4-acetyl- piperazin- 1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4- (dimethyl- carbamoyl) piperazine-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 2-(tert- butoxy- carbonyl- amino)CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H amino CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H 2- (methoxy- carbonyl- amino) CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H 4- methyl- sulfonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H bis (methoxy- carbonyl) amino CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H N-t- butoxy- carbonyl- N- methyl-amino CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H methyl- amino CH₂CH₂

C H H H 6-methyl- pyridin-3- yl CH₃ 4- methoxy- carbonyl- piperazin-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl CH₃ 4-t- butoxy- carbonyl- piperazin-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl CH₃ 4-acetyl- piperazin- 1-yl CH₂

C H H H 6-methyl- pyridin-3- yl CH₃ 4- methyl- sulfonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H acetyl- amino CH₂CH₂

C H H H 6-methyl- pyridin-3- yl CH₃ 4- (dimethyl- carbamoyl) piperazine-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H N,N- dimethyl- sulfamoyl- amino CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H 3,3- dimethyl- ureido CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H 4- methoxy- carbonyl- piperazin- 1-ylbond

C H H H 6-methyl- pyridin-3- yl H methoxy- carbonyl (methyl) aminoCH₂CH₂

C H H H 6-methyl- pyridin-3- yl H N- methyl- ethyl- sulfonamido CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H (N,N- dimethyl- sulfamoyl) (methyl)amino CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H 1,3,3- trimethyl ureido CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H N-acetyl- N-methyl- amino CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H benzyloxy- carbonyl amino CH₂CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H N,N- diethyl- amino CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H N,N- dimethyl- sulfamoyl- aminoCH₂CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H ethyl- sulfonamido CH₂CH₂CH₂

C H H H 6-methyl- pyridin-3- yl H 4- methoxy- carbonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H 4-t- butoxy- carbonyl- piperazin- 1-ylbond

C H H H 6-methyl- pyridin-3- yl H piperazin- 1-yl bond

C H H H 6-methyl- pyridin-3- yl H 4-acetyl- piperazin- 1-yl bond

C H H H 6-methyl- pyridin-3- yl H 4-(dimethyl- carbamoyl) piperazine-1-yl bond

C H H H 6-methyl- pyridin-3- yl H 4- methyl- sulfonyl- piperazin- 1-ylbond

C H H H 6-methyl- pyridin-3- yl H 4-t- butoxy- carbonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H 4- methoxy- carbonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H 4- ethoxy- carbonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H 4-acetyl- piperazin- 1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4-(dimethyl- carbamoyl) piperazine-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4- ethyl- sulfonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H 4-(N,N- dimethyl- sulfamoyl)piperazine- 1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4-t- butoxy- carbonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H 4-acetyl- piperazin- 1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4-(dimethyl- carbamoyl) piperazine-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4- methyl- sulfonyl- piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H 4- methoxy- carbonyl- piperazin- 1-ylCH₂

N H H H 6-methyl- pyridin-3- yl H 4- methoxy- carbonyl- piperazin- 1-ylCH₂

C H H H pyridin-3- yl H 4-t- butoxy- carbonyl- piperazin- 1-yl CH₂

N H H H 6-methyl- pyridin-3- yl H 4-acetyl- piperazin- 1-yl CH₂

N H H H 6-methyl- pyridin-3- yl H 4- methoxy- carbonyl- piperazin- 1-ylCH₂

N H H H 6-methoxy- pyridin-3- yl H 4-(4- acetyl- piperazine- 1-carbonyl)piperazin- 1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4-(4- methyl- piperazine- 1-carbonyl)piperazin- 1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4- (piperidine- 1-carbonyl) piperazin-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4- (morpholine- 4-carbonyl) piperazin-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4- (cyclobutyl- sulfonyl) piperazin-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4- (ethyl- sulfonyl) piperazin- 1-ylCH₂

C H H H 6-methyl- pyridin-3- yl H 4- (isopropyl- sulfonyl) piperazin-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4- (cyclopropyl- sulfonyl) piperazin-1-yl CH₂

C H H H 6-methyl- pyridin-3- yl H 4-(1,1- dioxide thio- morpholine-4-carbonyl) piperazin- 1-yl CH₂

C H H H 6-methyl- pyridin-3- yl

TABLE II

R⁶ R⁵ L A W¹ R¹ R² R³ R⁴ H 4- methoxy- carbonyl- piperazin-1-yl bond

C H H H 6- methyl-pyridin- 3-yl H 4- ethylsul- fonyl- piperazin-1-ylbond

C H H H 6- methyl-pyridin- 3-yl H 4-(N,N- dimethyl- sulfamoyl)piperazine-1-yl bond

C H H H 6- methyl-pyridin- 3-yl

and pharmaceutically acceptable salts, solvates, chelates, non-covalentcomplexes, prodrugs, and mixtures thereof.

Chemical entities of the invention can be synthesized utilizingtechniques well known in the art, e.g., as illustrated below withreference to the Reaction Schemes.

Unless specified to the contrary, the reactions described herein takeplace at atmospheric pressure, generally within a temperature range from−10° C. to 110° C. Further, except as employed in the Examples or asotherwise specified, reaction times and conditions are intended to beapproximate, e.g., taking place at about atmospheric pressure within atemperature range of about −10° C. to about 110° C. over a period ofabout 1 to about 24 hours; reactions left to run overnight average aperiod of about 16 hours.

The terms “solvent”, “organic solvent” or “inert solvent” each mean asolvent inert under the conditions of the reaction being described inconjunction therewith [including, for example, benzene, toluene,acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”),chloroform, methylene chloride (or dichloromethane), diethyl ether,methanol, pyridine and the like]. Unless specified to the contrary, thesolvents used in the reactions of the present invention are inertorganic solvents.

The term “q.s.” means adding a quantity sufficient to achieve a statedfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

Isolation and purification of the chemical entities and intermediatesdescribed herein can be effected, if desired, by any suitable separationor purification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, or a combination of these procedures.Specific illustrations of suitable separation and isolation procedurescan be had by reference to the examples hereinbelow. However, otherequivalent separation or isolation procedures can, of course, also beused.

When desired, the (R)- and (S)-isomers may be resolved by methods knownto those skilled in the art, for example by formation ofdiastereoisomeric salts or complexes which may be separated, forexample, by crystallization; via formation of diastereoisomericderivatives which may be separated, for example, by crystallization,gas-liquid or liquid chromatography; selective reaction of oneenantiomer with an enantiomer-specific reagent, for example enzymaticoxidation or reduction, followed by separation of the modified andunmodified enantiomers; or gas-liquid or liquid chromatography in achiral environment, for example on a chiral support, such as silica witha bound chiral ligand or in the presence of a chiral solvent. Forexample, a compound of Formula I can be dissolved in a lower alkanol andplaced on a Chiralpak AD (205×20 mm) column (Chiral Technologies, Inc.)conditioned for 60 min at 70% EtOAc in Hexane. It will be appreciatedthat where the desired enantiomer is converted into another chemicalentity by one of the separation procedures described above, a furtherstep may be required to liberate the desired enantiomeric form.Alternatively, a specific enantiomer may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer to the other by asymmetrictransformation.

It will be appreciated by those skilled in the art that one or more ofthe reactants, steps and/or conditions described in the reaction schemesmay require adjustment to accommodate various substituents at R₁ and R₂.

Many of the optionally substituted starting compounds 101a, 101b, 103,201, 301a and 301b and other reactants are commercially available, e.g.,from Aldrich Chemical Company (Milwaukee, Wis.) or can be readilyprepared by those skilled in the art using commonly employed syntheticmethodology.

Preparation of Compounds of Formula Ia and/or Ib

Referring to Reaction Scheme 1, a flask equipped with a magneticstirrer, reflux condenser and thermal well, under nitrogen, is chargedwith phosgene or a phosgene equivalent (typically triphosgene) and anonpolar, aprotic solvent such as dichloromethane or tetrahydrofuran. Asolution of a compound of Formula 101a or 101b in a nonpolar, aproticsolvent such as dichloromethane or tetrahydrofuran is added dropwiseover about 10-60 minutes and the solution is allowed to stir between 1to 15 hr. A compound of Formula 103 is added portionwise, and thesolution is stirred for about 10-60 min. A base, such as DIEA, is addeddropwise for about one hour, and the solution is allowed to stir forabout 1-15 hr. The product, a compound of Formula Ia and/or Ib, isisolated and purified.

Preparation of Compounds of Formula Ia and/or Ib

Reaction Scheme 2 illustrates an alternative synthesis of compounds ofFormula Ia and/or Ib. The isocyanate of Formula 201 can be formed andisolated independently from either corresponding amine (i.e., R⁴—NH₂)using phosgene or a phosgene equivalent or from the correspondingcarboxylic acid (i.e., R⁴—COOH) using a Curtius or Hoffmanrearrangement. A mixture of compounds of Formula 101a (or 101b) and 201in an aprotic solvent such as dichloromethane or tetrahydrofuran from−40° C. to 110° C. is allowed to stir for between 1 to 15 hr. Theproduct, a compound of Formula Ia (or Ib), is isolated and purified.

Preparation of Formula 303

Referring to Reaction Scheme 3, Step 1a, a compound of Formula 301a iscombined with about one equivalent of a compound of the formula R⁵—OHwherein R⁵ is as described above; a base such as potassium carbonate inan aprotic solvent such as DMF. The mixture is heated for about 1-16 hrat about 100° C. The product, a compound of Formula 303, is isolated andpurified.

Alternatively, as in Scheme 3, Step 1b, a compound of Formula 301b iscombined a compound of the formula R⁵—OH. The mixture is stirred about1-16 hr at about room temperature. The product, a compound of Formula303, is isolated and purified. Alternatively, as in Scheme 3, Step 1b, acompound of Formula 301b is treated with a base such as sodium hydridein an aprotic solvent such as DMF for 1-16 hours from 0° C. to 110° C. Acompound of the formula R⁵-Q wherein R⁵ is as described above and Q is aleaving group such as a halogen, methanesulfonate, a p-toluenesulfonate,or a trifluoromethanesulfonate in an aprotic solvent such as DMF or THFfor 1-16 hours from 0° C. to 110° C. The product, a compound of Formula303, is isolated and purified.

Preparation of Formula 305

Referring to Reaction Scheme 3, Step 2, a Parr hydrogenation bomb ischarged with 10% Pd/C under a nitrogen atmosphere, followed by asolution of a compound of Formula 303 in a polar, protic solvent such asethanol. The reaction is stirred for about 24 hr under about 70 psi H₂.The reaction mixture is filtered through celite and concentrated invacuo to afford a compound of Formula 305, which can be carried forwardto Formula I as illustrated with respect to Reaction Schemes 1 and 2.

Preparation of Regioisomer

Steps 1a, 1b and 2 can also be used with compounds where R³ and the NO₂group on the benzene ring are switched.

While it is well known that pharmaceuticals must meet pharmacopoeiastandards before approval and/or marketing, and that synthetic reagents(such as the various substituted amines or alcohols) and precursorsshould not exceed the limits prescribed by pharmacopoeia standards,final chemical entities prepared by a process of the present inventionmay have minor, but detectable, amounts of such materials present, forexample at levels in the range of 95% purity with no single impuritygreater than 1%. These levels can be detected, e.g., by emissionspectroscopy. It is important to monitor the purity of pharmaceuticalchemical entities for the presence of such materials, which presence isadditionally disclosed as a method of detecting use of a syntheticprocess of the invention.

A racemic mixture of isomers of a compound of Formula I is optionallyplaced on a chiral chromatography column and separated into (R)- and(S)-enantiomers.

A compound of Formula I is optionally contacted with a pharmaceuticallyacceptable acid to form the corresponding acid addition salt.

A pharmaceutically acceptable acid addition salt of Formula I isoptionally contacted with a base to form the corresponding free base ofFormula I.

The chemical entities of the present invention are selective for andmodulate the cardiac sarcomere, and are useful to bind to and/orpotentiate the activity of cardiac myosin, increasing the rate at whichmyosin hydrolyzes ATP. As used in this context, “modulate” means eitherincreasing or decreasing myosin activity, whereas “potentiate” means toincrease activity. It has also been determined in testing representativechemical entities of the invention, that their administration can alsoincrease the contractile force in cardiac muscle fiber.

The chemical entities, pharmaceutical compositions and methods of theinvention are used to treat heart disease, including but not limited to:acute (or decompensated) congestive heart failure, and chroniccongestive heart failure; for example, diseases associated with systolicheart dysfunction. Additional therapeutic utilities includeadministration to stabilize heart function in patients awaiting a hearttransplant, and to assist a stopped or slowed heart in resuming normalfunction following use of a bypass pump.

ATP hydrolysis is employed by myosin in the sarcomere to produce force.Therefore, an increase in ATP hydrolysis would correspond to an increasein the force or velocity of muscle contraction. In the presence ofactin, myosin ATPase activity is stimulated >100 fold. Thus, ATPhydrolysis not only measures myosin enzymatic activity but also itsinteraction with the actin filament. A chemical entity that modulatesthe cardiac sarcomere can be identified by an increase or decrease inthe rate of ATP hydrolysis by myosin, for example exhibiting a 1.4 foldincrease at concentrations less than 10 μM (for example, less than 1μM). Some assays for such activity will employ myosin from a humansource, although myosin from other organisms can also be used. Systemsthat model the regulatory role of calcium in myosin binding are alsouseful.

Alternatively, a biochemically functional sarcomere preparation can beused to determine in vitro ATPase activity, for example, as described inU.S. Ser. No. 09/539,164, filed Mar. 29, 2000. The functionalbiochemical behavior of the sarcomere, including calcium sensitivity ofATPase hydrolysis, can be reconstituted by combining its purifiedindividual components (including its regulatory components and myosin).Another functional preparation is the in vitro motility assay. It can beperformed by adding test chemical entity to a myosin-bound slide andobserving the velocity of actin filaments sliding over the myosincovered glass surface (Kron S J. (1991) Methods Enzymol. 196:399-416).

The in vitro rate of ATP hydrolysis correlates to myosin potentiatingactivity, which can be determined by monitoring the production of eitherADP or phosphate, for example as described in Ser. No. 09/314,464, filedMay 18, 1999. ADP production can also be monitored by coupling the ADPproduction to NADH oxidation (using the enzymes pyruvate kinase andlactate dehydrogenase) and monitoring the NADH level either byabsorbance or fluorescence (Greengard, P., Nature 178 (Part 4534):632-634 (1956); Mol Pharmacol 1970 January; 6(1):31-40). Phosphateproduction can be monitored using purine nucleoside phosphorylase tocouple phosphate production to the cleavage of a purine analog, whichresults in either a change in absorbance (Proc Natl Acad Sci USA 1992Jun. 1; 89(11):4884-7) or fluorescence (Biochem J 1990 Mar. 1;266(2):611-4). While a single measurement can be employed, multiplemeasurements may be taken of the same sample at different times in orderto determine the absolute rate of the protein activity; suchmeasurements can have higher specificity in the presence of testchemical entities that have similar absorbance or fluorescenceproperties with those of the enzymatic readout.

Test chemical entities can be assayed in a highly parallel fashion usingmultiwell plates by placing the chemical entities either individually inwells or testing them in mixtures. Assay components including the targetprotein complex, coupling enzymes and substrates, and ATP can then beadded to the wells and the absorbance or fluorescence of each well ofthe plate can be measured with a plate reader.

In one embodiment a 384 well plate format and a 25 μL reaction volume isused. A pyruvate kinase/lactate dehydrogenase coupled enzyme system(Huang T G and Hackney D D. (1994) J Biol Chem 269(23):16493-16501) isused to measure the rate of ATP hydrolysis in each well. As will beappreciated by those in the art, the assay components are added inbuffers and reagents. Since the methods outlined herein allow kineticmeasurements, incubation periods are optimized to give adequatedetection signals over the background. The assay is done in real timegiving the kinetics of ATP hydrolysis, which increases the signal tonoise ratio of the assay.

Modulation of cardiac muscle fiber contractile force can be measuredusing detergent permeabilized cardiac fibers (also referred to asskinned cardiac fibers), for example, as described by Haikala H, et al(1995) J Cardiovasc Pharmacol 25(5):794-801. Skinned cardiac fibersretain their intrinsic sarcomeric organization, but do not retain allaspects of cellular calcium cycling, this model offers two advantages:first, the cellular membrane is not a barrier to chemical entitypenetration, and second, calcium concentration is controlled. Therefore,any increase in contractile force is a direct measure of the testchemical entity's effect on sarcomeric proteins. Tension measurementsare made by mounting one end of the muscle fiber to a stationary postand the other end to a transducer that can measure force. Afterstretching the fiber to remove slack, the force transducer recordsincreased tension as the fiber begins to contract. This measurement iscalled the isometric tension, since the fiber is not allowed to shorten.Activation of the permeabilized muscle fiber is accomplished by placingit in a buffered calcium solution, followed by addition of test chemicalentity or control. When tested in this manner, chemical entities of theinvention caused an increase in force at calcium concentrationsassociated with physiologic contractile activity, but very littleaugmentation of force in relaxing buffer at low calcium concentrationsor in the absence of calcium (the EGTA data point).

Selectivity for the cardiac sarcomere and cardiac myosin can bedetermined by substituting non-cardiac sarcomere components and myosinin one or more of the above-described assays and comparing the resultsobtained against those obtained using the cardiac equivalents.

A chemical entity's ability to increase observed ATPase rate in an invitro reconstituted sarcomere assay could result from the increasedturnover rate of S1-myosin or, alternatively, increased sensitivity of adecorated actin filament to Ca⁺⁺-activation. To distinguish betweenthese two possible modes of action, the effect of the chemical entity onATPase activity of S1 with undecorated actin filaments is initiallymeasured. If an increase of activity is observed, the chemical entity'seffect on the Ca-responsive regulatory apparatus could be disproved. Asecond, more sensitive assay can be employed to identify chemicalentities whose activating effect on S1-myosin is enhanced in thepresence of a decorated actin (compared to pure actin filaments). Inthis second assay activities of cardiac-S1 and skeletal-S1 on cardiacand skeletal regulated actin filaments (in all 4 permutations) arecompared. A chemical entity that displays its effect oncardiac-S1/cardiac actin and cardiac-S1/skeletal actin, but not onskeletal-S1/skeletal actin and skeletal-S1/cardiac actin systems, can beconfidently classified as cardiac-S1 activator.

Initial evaluation of in vivo activity can be determined in cellularmodels of myocyte contractility, e.g., as described by Popping S, et al((1996) Am. J. Physiol. 271: H357-H364) and Wolska B M, et al ((1996)Am. J. Physiol. 39:H24-H32). One advantage of the myocyte model is thatthe component systems that result in changes in contractility can beisolated and the major site(s) of action determined. Chemical entitieswith cellular activity (for example, selecting chemical entities havingthe following profile: >120% increase in fractional shortening overbasal at 2 μM, limited changes in diastolic length (<5% change), and nosignificant decrease in contraction or relaxation velocities) can thenbe assessed in whole organ models, such as such as the Isolated Heart(Langendorff) model of cardiac function, in vivo using echocardiographyor invasive hemodynamic measures, and in animal-based heart failuremodels, such as the Rat Left Coronary Artery Occlusion model.Ultimately, activity for treating heart disease is demonstrated inblinded, placebo-controlled, human clinical trials.

At least one chemical entity as described herein is administered at atherapeutically effective dosage, e.g., a dosage sufficient to providetreatment for the disease states previously described. While humandosage levels have yet to be optimized for the chemical entities of theinvention, generally, a daily dose is from about 0.05 to 100 mg/kg ofbody weight, for example about 0.10 to 10.0 mg/kg of body weight, or,for example, about 0.15 to 1.0 mg/kg of body weight. Thus, foradministration to a 70 kg person, the dosage range would be about 3.5 to7000 mg per day, for example, about 7.0 to 700.0 mg per day, or forexample, about 10.0 to 100.0 mg per day. The amount of active chemicalentity administered will, of course, be dependent on the subject anddisease state being treated, the severity of the affliction, the mannerand schedule of administration and the judgment of the prescribingphysician; for example, a likely dose range for oral administrationwould be about 70 to 700 mg per day, whereas for intravenousadministration a likely dose range would be about 700 to 7000 mg perday, the active agents being selected for longer or shorter plasmahalf-lives, respectively.

Administration of the chemical entities of the invention or thepharmaceutically acceptable salts thereof can be via any of the acceptedmodes of administration for agents that serve similar utilitiesincluding, but not limited to, orally, subcutaneously, intravenously,intranasally, topically, transdermally, intraperitoneally,intramuscularly, intrapulmonarilly, vaginally, rectally, orintraocularly. Oral and parenteral administration are customary intreating the indications that are the subject of the present invention.

Pharmaceutically acceptable compositions include solid, semi-solid,liquid and aerosol dosage forms, such as, e.g., tablets, capsules,powders, liquids, suspensions, suppositories, aerosols or the like. Thechemical entities can also be administered in sustained or controlledrelease dosage forms, including depot injections, osmotic pumps, pills,transdermal (including electrotransport) patches, and the like, forprolonged and/or timed, pulsed administration at a predetermined rate.In certain embodiments, the compositions are provided in unit dosageforms suitable for single administration of a precise dose.

The chemical entities can be administered either alone or more typicallyin combination with a conventional pharmaceutical carrier, excipient orthe like (e.g., mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin,sucrose, magnesium carbonate, and the like). If desired, thepharmaceutical composition can also contain minor amounts of nontoxicauxiliary substances such as wetting agents, emulsifying agents,solubilizing agents, pH buffering agents and the like (e.g., sodiumacetate, sodium citrate, cyclodextrine derivatives, sorbitanmonolaurate, triethanolamine acetate, triethanolamine oleate, and thelike). Generally, depending on the intended mode of administration, thepharmaceutical formulation will contain about 0.005% to 95%, or about0.5% to 50% by weight of a chemical entity of the invention. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

In addition, the chemical entities of the invention can beco-administered with, and the pharmaceutical compositions can include,other medicinal agents, pharmaceutical agents, adjuvants, and the like.Suitable additional active agents include, for example: therapies thatretard the progression of heart failure by down-regulating neurohormonalstimulation of the heart and attempt to prevent cardiac remodeling(e.g., ACE inhibitors or β-blockers); therapies that improve cardiacfunction by stimulating cardiac contractility (e.g., positive inotropicagents, such as the β-adrenergic agonist dobutamine or thephosphodiesterase inhibitor milrinone); and therapies that reducecardiac preload (e.g., diuretics, such as furosemide).

In one embodiment, the pharmaceutical compositions will take the form ofa pill or tablet and thus the composition will contain, along with theactive ingredient, a diluent such as lactose, sucrose, dicalciumphosphate, or the like; a lubricant such as magnesium stearate or thelike; and a binder such as starch, gum acacia, polyvinylpyrrolidine,gelatin, cellulose, cellulose derivatives or the like. In another soliddosage form, a powder, marume, solution or suspension (e.g., inpropylene carbonate, vegetable oils or triglycerides) is encapsulated ina gelatin capsule.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active chemical entity asdefined above and optional pharmaceutical adjuvants in a carrier (e.g.,water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like)to form a solution or suspension. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, asemulsions, or in solid forms suitable for dissolution or suspension inliquid prior to injection. The percentage of active chemical entitycontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the chemical entityand the needs of the subject. However, percentages of active ingredientof 0.01% to 10% in solution are employable, and will be higher if thecomposition is a solid that will be subsequently diluted to the abovepercentages. In certain embodiments, the composition will comprise0.2-2% of the active agent in solution.

Formulations of the active chemical entity or a salt may also beadministered to the respiratory tract as an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation have diameters of less than 50 microns, forexample, less than 10 microns.

Generally, to employ the chemical entities of the invention in a methodof screening for myosin binding, myosin is bound to a support and achemical entity of the invention is added to the assay. Alternatively,the chemical entity of the invention can be bound to the support and themyosin added. Classes of chemical entities among which novel bindingagents may be sought include specific antibodies, non-natural bindingagents identified in screens of chemical libraries, peptide analogs,etc. Of interest are screening assays for candidate agents that have alow toxicity for human cells. A wide variety of assays may be used forthis purpose, including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,functional assays (phosphorylation assays, etc.) and the like. See,e.g., U.S. Pat. No. 6,495,337, incorporated herein by reference.

The following examples serve to more fully describe the manner of usingthe above-described invention. It is understood that these examples inno way serve to limit the true scope of this invention, but rather arepresented for illustrative purposes. All references cited herein areincorporated by reference in their entirety.

EXAMPLES Example 1

4-Nitro-2,3-dihydro-1H-inden-1-one

To a 0° C. solution of indanone (1.1 mL, 9.16 mmol, 1.0 equiv) inconcentrated H2SO4 (9 mL) was added KNO3 (926 mg, 9.16 mmol, 1.0 equiv)as a solid in several portions over 5 min. After stirring for 1 h, thereaction mixture was poured onto ice. The aqueous suspension wasextracted with EtOAC (3×30 mL), and the combined organic layers werewashed with brine, dried over sodium sulfate, and concentrated in vacuo.Purification by silica gel chromatography (20%-50% EtOAc/Hexanes)provided the desired compound as a yellow solid (288 mg, 18%).

4-Nitro-2,3-dihydro-1H-inden-1-ol

To a 0° C. solution of 4-nitro-2,3-dihydro-1H-inden-1-one (149 mgs, 0.84mmol, 1.0 equiv) in MeOH (2 mL) was added NaBH4 (10 mg, 0.26 mmol, 0.33equiv) as a solid in one portion. After stirring for 1 h at 0° C., thesolvent was removed in vacuo, and water (3 mL) was added to the residue.The aqueous suspension was extracted three times with EtOAc, and thecombined organic layers were dried over sodium sulfate. The solution wasfiltered and concentrated in vacuo. The desired product was used withoutfurther purification (135 mg, 90%).

1-Chloro-4-nitro-2,3-dihydro-1H-indene

To a 0° C. solution of 4-nitro-2,3-dihydro-1H-inden-1-ol (252 mg, 1.41mmol, 1.0 equiv) in dry toluene (2.5 mL) was added thionyl chloride (160μL, 2.11 mmol, 1.5 equiv) dropwise by syringe. After stirring at 0° C.for 30 min, the reaction mixture was heated to 55° C. for 2 h. Thereaction was allowed to cool to room temperature, washed twice withwater, dried over sodium sulfate, filtered and concentrated in vacuo.The unpurified alkyl chloride (221 mg, 79%) was used without furtherpurification.

Methyl 4-(4-nitro-2,3-dihydro-1H-inden-1-yl)piperazine-1-carboxylate

To a room temperature solution of 1-Chloro-4-nitro-2,3-dihydro-1H-indene(221 mg, 1.12 mmol, 1.0 equiv) and DIPEA (430 μL, 2.46 mmol, 2.2 equiv)in dry acetonitrile (1.6 mL) was added methylpiperazine carboxylatehydrochloride salt (444 mg, 2.46 mmol, 2.2 equiv) as a solid in oneportion. The resulting reaction mixture was heated to 79° C. overnight.The reaction mixture was allowed to cool to RT and diluted with EtOAc.The organic layer was extracted with 1 M HCl (3×20 mL), and the combinedaqueous layers were treated with 3 N NaOH until the pH=10. The resultingsolution was extracted three times with EtOAc, and the combined organiclayers were dried over sodium sulfate, filtered and concentrated invacuo to provide the title compound as a brown oil (239 mg, 78%). LCMS[M+H]⁺=306.1.

Methyl 4-(4-amino-2,3-dihydro-1H-inden-1-yl)piperazine-1-carboxylate

To a room temperature solution of methyl4-(4-nitro-2,3-dihydro-1H-inden-1-yl)piperazine-1-carboxylate (216 mg,0.71 mmol, 1.0 equiv) in MeOHH (2.8 mL) and glacial HOAc (2.8 mL) wasadded iron powder (198 mg, 3.54 mmol, 5.0 equiv) as a solid in oneportion. After stirring overnight, the reaction mixture was concentratedin vacuo and then diluted with EtOAc and 3 N NaOH. The organic layer waswashed with brine and concentrated in vacuo to provide the titlecompound as a brown oil (180 mg, 92%).

Methyl4-(4-(3-(6-methylpyridin-3-yl)ureido)-2,3-dihydro-1H-inden-1-yl)piperazine-1-carboxylate

To a room temperature solution of Methyl4-(4-amino-2,3-dihydro-1H-inden-1-yl)piperazine-1-carboxylate (175 mg,0.635 mmol, 1.0 equiv) and triethylamine (90 μL, 0.635 mmol, 1.0 equiv)in dry DCM (1.7 mL) was added freshly filtered2-methyl-5-isocyanatopyridine (94 mg, 0.699 mmol, 1.1 equiv) in dry DCM(1.7 mL) dropwise via cannula. After 1 h, the reaction was diluted withDCM and washed with water and with brine. The resulting solution wasdried over sodium sulfate, filtered, and concentrated in vacuo. Thesolid was dissolved in a minimal volume of DMF and water was added toprecipitate the product. The resulting solid was filtered, washed threetimes with water and twice with hexane to provide the title compound asa light brown solid (133 mg, 51%). LCMS [M+H]⁺=410.1.

Example 2

2-(Chloromethyl)-4-nitrobenzo[d]oxazole

To a RT solution of 2-amino-3-nitrophenol (14.7 g, 95 mmol, 1.0 equiv)in 2-methoxyethylether (136 mL) was added 2-chloro-1,1,1-triethoxyethane(19.1 g, 97.3 mmol, 1.02 equiv) and p-toluene sulfonic acid hydrate (25mg, 0.13 mmol, 0.2 equiv). The resulting mixture was heate to refluxovernight. After the reaction was cooled to RT, the solvent was removedin vacuo. The solid was suspended in methanol (40 mL), stirred andfiltered to provide the desired product as a red solid (3.61 g). Thefiltrate provided an additional 9.8 g of the title compound through anadditional methanol treatment (13.4 g, 98% combined).

tert-Butyl4-((4-nitrobenzo[d]oxazol-2-yl)methyl)piperazine-1-carboxylate

To room temperature solution of 2-(Chloromethyl)-4-nitrobenzo[d]oxazole(5.15 g, 24.2 mmol, 1.0 equiv) and DIPEA (4.2 mL, 24.2 mmol, 1.0 equiv)in dry acetonitrile (119 mL) was added N-Boc piperazine as a solid inone portion. The reaction was stirred at room temperature overnight. Themixture was diluted with EtOAc (120 mL), washed with water and brine,dried over sodium sulfate, filtered and concentrated in vacuo.Purification on silica gel (20% EtOAc/80% hexanes—100% EtOAc) providedthe title compound as a yellow solid (7.0 g, 68%).

tert-Butyl4-((4-aminobenzo[d]oxazol-2-yl)methyl)piperazine-1-carboxylate

To a solution of tert-butyl4-((4-nitrobenzo[d]oxazol-2-yl)methyl)piperazine-1-carboxylate (5.97 g,16.6 mmol, 1.0 equiv) in MeOH (49 mL) was added Pd/C (10% Pd, wet, 2.99g). The resulting suspension was fixed with a hydrogen balloon andsparged with hydrogen while stirring was maintained. After 1 h, themixture was filtered through a pad of celite and concentrated in vacuoto provide the title compound as an off-white solid (5.0 g, 91%).

tert-Butyl4-((4-(3-(6-methylpyridin-3-yl)ureido)benzo[d]oxazol-2-yl)methyl)piperazine-1-carboxylate

To a room temperature solution of tert-butyl4-((4-aminobenzo[d]oxazol-2-yl)methyl)piperazine-1-carboxylate (5.0 g,15.1 mmol, 1.0 equiv) and triethylamine (2.1 g, 15.1 mmol, 1.0 equiv) indry DCM (37 mL) was added 2-methyl-5-isocyanatopyridine (2.22 g, 16.6mmol, 1.1 equiv) in dry DCM (37 mL) via cannula. After 1 h, the reactionmixture was filtered and washed with water (25 mL) and brine (25 mL).The solution was dried over sodium sulfate, filtered and concentrated invacuo. Silica gel purification (5% MeOH/95% EtOAc—15% MeOH/85% EtOAc)provided the title compound as a white foam (7.03 g, 100%). LCMS[M+H]⁺=467.2.

To a room temperature solution of tert-butyl4-((4-(3-(6-methylpyridin-3-yl)ureido)benzo[d]oxazol-2-yl)methyl)piperazine-1-carboxylate(7.0 g, 15 mmol, 1.0 equiv) in MeOH (295 mL) was added HCl (4.0 M indioxane, 75 mL, 300 mmol, 20 equiv) by syringe. After 3 h, solvents wereremoved in vacuo and the resulting solid was used without furtherpurification. A portion of the resulting deprotected amine salt (541 mg,1.14 mmol, 1.0 equiv) was suspended in dry DCM (10 mL), and DIPEA (890uL, 5.12 mmol, 4.5 equiv) was added. To this mixture was added dimethylcarbamoyl chloride (130 uL, 1.36 mmol, 1.2 equiv) and the reaction wasstirred overnight. The reaction was washed with water and brine, driedover sodium sulfate, and concentrated in vacuo. Purification by silicagel chromatography (2% MeOH/98% EtOAc—10% MeOH/90% EtOAc) provided thetitle compound as a white foam (428 mg, 94%). LCMS [M+H]⁺=438.1.

Example 3

Following procedures similar to those described herein, the followingcompounds were prepared:

Compound Mass Spec methyl4-(5-{[(6-methyl-3-pyridyl)amino]carbonylamino}- 424 (M + H)+1,2,3,4-tetrahydronaphthyl)piperazinecarboxylate tert-butyl4-[(4-{[(6-methyl-3- pyridyl)amino]carbonylamino}benzoxazol-2-yl)methyl]piperazinecarboxylate methyl 4-[(4-{[(6-methyl-3- 398 (M + H)pyridyl)amino]carbonylamino}benzoxazol-2-yl)methyl]piperazinecarboxylateN-{2-[(4-acetylpiperazinyl)methyl]benzoxazol-4-yl}[(6-methyl(3-pyridyl))amino]carboxamide N-(2-{[4-(N,N-dimethylcarbamoyl)piperazinyl]methyl}benzoxazol-4-yl)[(6-methyl(3-pyridyl))amino]carboxamide (tert-butoxy)-N-[2-(5-{[(6-methyl(3-396 (M + H) pyridyl))amino]carbonylamino}(2-1,2,3,4-tetrahydroisoquinolyl))ethyl]carboxamideN-[2-(2-aminoethyl)(5-1,2,3,4-tetrahydroisoquinolyl)][(6- 326 (M + H)methyl(3-pyridyl))amino]carboxamide methoxy-N-[2-(5-{[(6-methyl(3- 384(M + H) pyridyl))amino]carbonylamino}(2-1,2,3,4-tetrahydroisoquinolyl))ethyl]carboxamide[(6-methyl(3-pyridyl))amino]-N-(2-{[4-(methylsulfonyl)piperazinyl]methyl}benzoxazol-4- yl)carboxamide methyl{methoxy-N-[2-(5-{[(6-methyl(3- 442 (M + H)pyridyl))amino]carbonylamino}(2-1,2,3,4-tetrahydroisoquinolyl))ethyl]carbonylamino}formate(tert-butoxy)-N-methyl-N-[2-(5-{[(6-methyl(3- 440 (M + H)pyridyl))amino]carbonylamino}(2-1,2,3,4-tetrahydroisoquinolyl))ethyl]carboxamide[(6-methyl(3-pyridyl))amino]-N-{2-[2-(methylamino)ethyl](5- 340 (M + H)1,2,3,4-tetrahydroisoquinolyl)}carboxamide tert-butyl4-[(1-methyl-7-{[(6-methyl(3- pyridyl))amino]carbonylamino}indol-2-yl)methyl]piperazinecarboxylate[(6-methyl(3-pyridyl))amino]-N-(1-methyl-2-{[4-(methylsulfonyl)piperazinyl]methyl}indol-7-yl)carboxamideN-[2-(5-{[(6-methyl-3-pyridyl)amino]carbonylamino}-2-1,2,3,4-tetrahydroisoquinolyl)ethyl]acetamideN,N-dimethyl{4-[(1-methyl-7-{[(6-methyl(3-pyridyl))amino]carbonylamino}indol-2- yl)methyl]piperazinyl}carboxamideN-[2-(2-{[(dimethylamino)sulfonyl]amino}ethyl)(5-1,2,3,4- 433 (M + H)tetrahydroisoquinolyl)][(6-methyl(3-pyridyl))amino]carboxamide methyl4-(4-{[(6-methyl-3- 410 (M + H)+pyridyl)amino]carbonylamino}indanyl)piperazinecarboxylatemethoxy-N-methyl-N-[2-(5-{[(6-methyl(3- 398 (M + H)pyridyl))amino]carbonylamino}(2-1,2,3,4-tetrahydroisoquinolyl))ethyl]carboxamideN-(2-{2-[(ethylsulfonyl)methylamino]ethyl}(5-1,2,3,4- 432 (M + H)tetrahydroisoquinolyl))[(6-methyl(3-pyridyl))amino]carboxamideN-[2-(2-{[(dimethylamino)sulfonyl]methylamino}ethyl)(5- 447 (M + H)1,2,3,4-tetrahydroisoquinolyl)][(6-methyl(3- pyridyl))amino]carboxamide(dimethylamino)-N-methyl-N-[2-(5-{[(6-methyl(3- 411 (M + H)pyridyl))amino]carbonylamino}(2-1,2,3,4-tetrahydroisoquinolyl))ethyl]carboxamide N-methyl-N-[2-(5-{[(6-methyl(3-382 (M + H) pyridyl))amino]carbonylamino}(2-1,2,3,4-tetrahydroisoquinolyl))ethyl]acetamide[(6-methyl(3-pyridyl))amino]-N-(2-{3- 474 (M + H)[(phenylmethoxy)carbonylamino]propyl}(5-1,2,3,4-tetrahydroisoquinolyl))carboxamideN-{2-[2-(diethylamino)ethyl](5-1,2,3,4- 382 (M + H)tetrahydroisoquinolyl)}[(6-methyl(3-pyridyl))amino]carboxamideN-[2-(3-{[(dimethylamino)sulfonyl]amino}propyl)(5-1,2,3,4- 447 (M + H)tetrahydroisoquinolyl)][(6-methyl(3-pyridyl))amino]carboxamideN-(2-{3-[(ethylsulfonyl)amino]propyl}(5-1,2,3,4- 432 (M + H)tetrahydroisoquinolyl))[(6-methyl(3-pyridyl))amino]carboxamide methyl4-(6-{[(6-methyl-3-pyridyl)amino]carbonylamino}indanyl)piperazinecarboxylateN-{3-[4-(ethylsulfonyl)piperazinyl]indan-5-yl}[(6-methyl(3-pyridyl))amino]carboxamideN-(3-{4-[(dimethylamino)sulfonyl]piperazinyl}indan-5-yl)[(6-methyl(3-pyridyl))amino]carboxamide methyl4-[(7-{[(6-methyl-3-pyridyl)amino]carbonylamino}indol-2-yl)methyl]piperazinecarboxylate tert-butyl 4-(4-{[(6-methyl-3- 452(M + H)+ pyridyl)amino]carbonylamino}indanyl)piperazinecarboxylate[(6-methyl(3-pyridyl))amino]-N-(1-piperazinylindan-4- 352 (M + H)+yl)carboxamide N-[1-(4-acetylpiperazinyl)indan-4-yl][(6-methyl(3- 394(M + H)+ pyridyl))amino]carboxamideN-{1-[4-(N,N-dimethylcarbamoyl)piperazinyl]indan-4-yl}[(6- 423 (M + H)+methyl(3-pyridyl))amino]carboxamide[(6-methyl(3-pyridyl))amino]-N-{1-[4- 430 (M + H)+(methylsulfonyl)piperazinyl]indan-4-yl}carboxamide tert-butyl4-[(4-{[(6-methyl-3- 466 (M + H+) pyridyl)amino]carbonylamino}indan-2-yl)methyl]piperazinecarboxylate methyl4-[(4-{[(6-methyl-3-pyridyl)amino]carbonylamino}indan- 424 (M + H+)2-yl)methyl]piperazinecarboxylate ethyl4-[(4-{[(6-methyl-3-pyridyl)amino]carbonylamino}indan-2- 438 (M + H+)yl)methyl]piperazinecarboxylateN-{2-[(4-acetylpiperazinyl)methyl]indan-4-yl}[(6-methyl(3- 408 (M + H+)pyridyl))amino]carboxamideN-(2-{[4-(N,N-dimethylcarbamoyl)piperazinyl]methyl}indan-4- 437 (M + H+)yl)[(6-methyl(3-pyridyl))amino]carboxamideN-(2-{[4-(ethylsulfonyl)piperazinyl]methyl}indan-4-yl)[(6- 458 (M + H+)methyl(3-pyridyl))amino]carboxamideN-[2-({4-[(dimethylamino)sulfonyl]piperazinyl}methyl)indan-4- 474 (M +H+) yl][(6-methyl(3-pyridyl))amino]carboxamide tert-butyl4-[(7-{[(6-methyl-3- pyridyl)amino]carbonylamino}indol-2-yl)methyl]piperazinecarboxylateN-{2-[(4-acetylpiperazinyl)methyl]indol-7-yl}[(6-methyl(3-pyridyl))amino]carboxamideN-{2-[(4-acetylpiperazinyl)methyl]indol-7-yl}[(6-methyl(3-pyridyl))amino]carboxamideN-(2-{[4-(N,N-dimethylcarbamoyl)piperazinyl]methyl}indol-7-yl)[(6-methyl(3-pyridyl))amino]carboxamide[(6-methyl(3-pyridyl))amino]-N-(2-{[4-(methylsulfonyl)piperazinyl]methyl}indol-7-yl)carboxamide methyl4-[(8-{[(6-methyl-3-pyridyl)amino]carbonylamino}-4-hydroimidazo[1,2-a]pyridin-2-yl)methyl]piperazinecarboxylate tert-butyl4-[(8-{[(6-methyl-3-pyridyl)amino]carbonylamino}-4-hydroimidazo[1,2-a]pyridin-2-yl)methyl]piperazinecarboxylateN-{2-[(4-acetylpiperazinyl)methyl](4-hydroimidazo[1,2-a]pyridin-8-yl)}[(6-methyl(3-pyridyl))amino]carboxamide methyl4-[(8-{[(6-methoxy-3-pyridyl)amino]carbonylamino}-4-hydroimidazo[1,2-a]pyridin-2-yl)methyl]piperazinecarboxylateN-(2-{[4-(azetidinylsulfonyl)piperazinyl]methyl}benzoxazol-4-yl)[(6-methyl(3-pyridyl))amino]carboxamideN-(2-{[4-(ethylsulfonyl)piperazinyl]methyl}benzoxazol-4-yl)[(6-methyl(3-pyridyl))amino]carboxamide[(6-methyl(3-pyridyl))amino]-N-[2-({4-[(methylethyl)sulfonyl]piperazinyl}methyl)benzoxazol-4- yl]carboxamideN-(2-{[4-(cyclopropylsulfonyl)piperazinyl]methyl}benzoxazol-4-yl)[(6-methyl(3-pyridyl))amino]carboxamideN-(2-{[4-(azetidinylcarbonyl)piperazinyl]methyl}benzoxazol-4-yl)[(6-methyl(3-pyridyl))amino]carboxamide[(6-methyl(3-pyridyl))amino]-N-(2-{[4-(morpholin-4-ylcarbonyl)piperazinyl]methyl}benzoxazol-4-yl)carboxamide[(6-methyl(3-pyridyl))amino]-N-[2-({4-[(4-methylpiperazinyl)carbonyl]piperazinyl}methyl)benzoxazol-4-yl]carboxamide [(6-methyl(3-pyridyl))amino]-N-(2-{[4-(pyrrolidinylcarbonyl)piperazinyl]methyl}benzoxazol-4- yl)carboxamide[(6-methyl(3-pyridyl))amino]-N-(2-{[4-(piperidylcarbonyl)piperazinyl]methyl}benzoxazol-4- yl)carboxamideN-[2-({4-[(1,1-dioxo(1,4-thiazaperhydroin-4-yl))carbonyl]piperazinyl}methyl)benzoxazol-4-yl][(6-methyl(3-pyridyl))amino]carboxamide N-[2-({4-[(4-acetylpiperazinyl)carbonyl]piperazinyl}methyl)benzoxazol-4-yl][(6-methyl(3-pyridyl))amino]carboxamide tert-butyl4-((8-(3-(6-methylpyridin-3-yl)ureido)imidazo[1,2-a]pyridin-2-yl)methyl)piperazine-1-carboxylate1-(6-methylpyridin-3-yl)-3-(2-((4-(piperidine-1-carbonyl)piperazin-1-yl)methyl)benzo[d]oxazol-4-yl)urea methyl4-((1-methyl-7-(3-(6-methylpyridin-3-yl)ureido)-1H-indol-2-yl)methyl)piperazine-1-carboxylate

Example 4 Target Identification Assays

Specificity Assays:

Specificity towards cardiac myosin is evaluated by comparing the effectof the chemical entity on actin-stimulated ATPase of a panel of myosinisoforms: cardiac, skeletal and smooth muscle, at a single 50 μMconcentration or to multiple concentrations of the chemical entity.

Myofibril Assays:

To evaluate the effect of compounds on the ATPase activity offull-length cardiac myosin in the context of native sarcomere, skinnedmyofibril assays are performed. Rat cardiac myofibrils are obtained byhomogenizing rat cardiac tissue in the presence of detergent. Suchtreatment removes membranes and majority of soluble cytoplasmic proteinsbut leaves intact cardiac sarcomeric acto-myosin apparatus. Myofibrilpreparations retain the ability to hydrolyze ATP in an Ca⁺⁺ controlledmanner. ATPase activities of such myofibril preparations in the presenceand absence of compounds are assayed at Ca⁺⁺ concentrations giving 50%and 100% of a maximal rate.

Example 5 In Vitro Model of Dose Dependent Cardiac Myosin ATPaseModulation

Dose responses are measured using a calcium-buffered, pyruvate kinaseand lactate dehydrogenase-coupled ATPase assay containing the followingreagents (concentrations expressed are final assay concentrations):Potassium PIPES (12 mM), MgCl₂ (2 mM), ATP (1 mM), DTT (1 mM), BSA (0.1mg/ml), NADH (0.5 mM), PEP (1.5 mM), pyruvate kinase (4 U/ml), lactatedehydrogenase (8 U/ml), and antifoam (90 ppm). The pH is adjusted to6.80 at 22° C. by addition of potassium hydroxide. Calcium levels arecontrolled by a buffering system containing 0.6 mM EGTA and varyingconcentrations of calcium, to achieve a free calcium concentration of1×10⁻⁴ M to 1×10⁻⁸ M.

The protein components specific to this assay are bovine cardiac myosinsubfragment-1 (typically 0.5 μM), bovine cardiac actin (14 μM), bovinecardiac tropomyosin (typically 3 μM), and bovine cardiac troponin(typically 3-8 μM). The exact concentrations of tropomyosin and troponinare determined empirically, by titration to achieve maximal differencein ATPase activity when measured in the presence of 1 mM EGTA versusthat measured in the presence of 0.2 mM CaCl₂. The exact concentrationof myosin in the assay is also determined empirically, by titration toachieve a desired rate of ATP hydrolysis. This varies between proteinpreparations, due to variations in the fraction of active molecules ineach preparation.

Chemical entity dose responses are typically measured at the calciumconcentration corresponding to 50% of maximal ATPase activity (pCa₅₀),so a preliminary experiment is performed to test the response of theATPase activity to free calcium concentrations in the range of 1×10⁻⁴ Mto 1×10⁻⁸ M. Subsequently, the assay mixture is adjusted to the pCa₅₀(typically 3×10⁻⁷ M). Assays are performed by first preparing a dilutionseries of test chemical entity, each with an assay mixture containingpotassium Pipes, MgCl₂, BSA, DTT, pyruvate kinase, lactatedehydrogenase, myosin subfragment-1, antifoam, EGTA, CaCl₂, and water.The assay is started by adding an equal volume of solution containingpotassium Pipes, MgCl₂, BSA, DTT, ATP, NADH, PEP, actin, tropomyosin,troponin, antifoam, and water. ATP hydrolysis is monitored by absorbanceat 340 nm. The resulting dose response curve is fit by the 4 parameterequation y=Bottom+((Top-Bottom)/(1+((EC50/X)^Hill))). The AC1.4 isdefined as the concentration at which ATPase activity is 1.4-fold higherthan the bottom of the dose curve.

Example 6 Myocyte Assays

A. Preparation of Adult Cardiac Ventricular Rat Myocytes.

Adult male Sprague-Dawley rats are anesthetized with a mixture ofisoflurane gas and oxygen. Hearts are quickly excised, rinsed and theascending aorta cannulated. Continuous retrograde perfusion is initiatedon the hearts at a perfusion pressure of 60 cm H₂O. Hearts are firstperfused with a nominally Ca²⁺ free modified Krebs solution of thefollowing composition: 110 mM NaCl, 2.6 mM KCL, 1.2 mM KH₂PO₄ 7H₂O, 1.2mM MgSO₄, 2.1 mM NaHCO₃, 11 mM glucose and 4 mM Hepes (all Sigma). Thismedium is not recirculated and is continually gassed with O₂. Afterapproximately 3 minutes the heart is perfused with modified Krebs buffersupplemented with 3.3% collagenase (169 μ/mg activity, Class II,Worthington Biochemical Corp., Freehold, N.J.) and 25 μM final calciumconcentration until the heart becomes sufficiently blanched and soft.The heart is removed from the cannulae, the atria and vessels discardedand the ventricles are cut into small pieces. The myocytes are dispersedby gentle agitation of the ventricular tissue in fresh collagenasecontaining Krebs prior to being gently forced through a 200 μm nylonmesh in a 50 cc tube. The resulting myocytes are resuspended in modifiedKrebs solution containing 25 μm calcium. Myocytes are made calciumtolerant by addition of a calcium solution (100 mM stock) at 10 minuteintervals until 100 μM calcium is achieved. After 30 minutes thesupernatant is discarded and 30-50 ml of Tyrode buffer (137 mM NaCL, 3.7mM KCL, 0.5 mM MgCL, 11 mM glucose, 4 mM Hepes, and 1.2 mM CaCl₂, pH7.4) is added to cells. Cells are kept for 60 min at 37° C. prior toinitiating experiments and used within 5 hrs of isolation. Preparationsof cells are used only if cells first passed QC criteria by respondingto a standard (>150% of basal) and isoproterenol (ISO; >250% of basal).Additionally, only cells whose basal contractility is between 3 and 8%are used in the following experiments.

B. Adult Ventricular Myocyte Contractility Experiments.

Aliquots of Tyrode buffer containing myocytes are placed in perfusionchambers (series 20 RC-27NE; Warner Instruments) complete with heatingplatforms. Myocytes are allowed to attach, the chambers heated to 37°C., and the cells then perfused with 37° C. Tyrode buffer. Myocytes arefield stimulated at 1 Hz in with platinum electrodes (20% abovethreshold). Only cells that have clear striations, and are quiescentprior to pacing are used for contractility experiments. To determinebasal contractility, myocytes are imaged through a 40× objective andusing a variable frame rate (60-240 Hz) charge-coupled device camera,the images are digitized and displayed on a computer screen at asampling speed of 240 Hz. [Frame grabber, myopacer, acquisition, andanalysis software for cell contractility are available from IonOptix(Milton, Mass.).] After a minimum 5 minute basal contractility period,test compounds (0.01-15 μM) are perfused on the myocytes for 5 minutes.After this time, fresh Tyrode buffer is perfused to determine compoundwashout characteristics. Using edge detection strategy, contractility ofthe myocytes and contraction and relaxation velocities are continuouslyrecorded.

C. Contractility Analysis:

Three or more individual myocytes are tested per compound, using two ormore different myocyte preparations. For each cell, twenty or morecontractility transients at basal (defined as 1 min prior to compoundinfusion) and after compound addition, are averaged and compared. Theseaverage transients are analyzed to determine changes in diastoliclength, and using the Ionwizard analysis program (IonOptix), fractionalshortening (% decrease in the diastolic length), and maximum contractionand relaxation velocities (um/sec) are determined. Analyses ofindividual cells are combined. Increase in fractional shortening overbasal indicates potentiation of myocyte contractility.

D. Calcium Transient Analysis:

Fura Loading:

Cell permeable Fura-2 (Molecular Probes) is dissolved in equal amountsof pluronic (Mol Probes) and FBS for 10 min at RT. A 1 μM Fura stocksolution is made in Tyrode buffer containing 500 mM probenecid (Sigma).To load cells, this solution is added to myocytes at RT. After 10 min.the buffer is removed, the cells washed with Tyrode containingprobenecid and incubated at RT for 10 min. This wash and incubation isrepeated. Simultaneous contractility and calcium measurements aredetermined within 40 min. of loading.

Imaging:

A test compound is perfused on cells. Simultaneous contractility andcalcium transient ratios are determined at baseline and after compoundaddition. Cells are digitally imaged and contractility determined asdescribed above, using that a red filter in the light path to avoidinterference with fluorescent calcium measurements. Acquisition,analysis software and hardware for calcium transient analysis areobtained from IonOptix. The instrumentation for fluorescence measurementincludes a xenon arc lamp and a Hyperswitch dual excitation light sourcethat alternates between 340 and 380 wavelengths at 100 Hz by agalvo-driven mirror. A liquid filled light guide delivers the dualexcitation light to the microscope and the emission fluorescence isdetermined using a photomultiplier tube (PMT). The fluorescence systeminterface routes the PMT signal and the ratios are recorded using theIonWizard acquisition program.

Analysis:

For each cell, ten or more contractility and calcium ratio transients atbasal and after compound addition, where averaged and compared.Contractility average transients are analyzed using the Ionwizardanalysis program to determine changes in diastolic length, andfractional shortening (% decrease in the diastolic length). The averagedcalcium ratio transients are analyzed using the Ionwizard analysisprogram to determine changes in diastolic and systolic ratios and the75% time to baseline (T₇₅).

E. Durability:

To determine the durability of response, myocytes are challenged with atest compound for 25 minutes followed by a 2 min. washout period.Contractility response is compared at 5 and 25 min. following compoundinfusion.

F. Threshold Potential:

Myocytes are field stimulated at a voltage approximately 20% abovethreshold. In these experiments the threshold voltage (minimum voltageto pace cell) is empirically determined, the cell paced at thatthreshold and then the test compound is infused. After the compoundactivity is at steady state, the voltage is decreased for 20 seconds andthen restarted. Alteration of ion channels corresponds to increasing orlowering the threshold action potential.

G. Hz Frequency:

Contractility of myocytes is determined at 3 Hz as follows: a 1 min.basal time point followed by perfusion of the test compound for 5 min.followed by a 2 min. washout. After the cell contractility has returnedcompletely to baseline the Hz frequency is decreased to 1. After aninitial acclimation period the cell is challenged by the same compound.As this species, rat, exhibits a negative force frequency at 1 Hz, at 3Hz the FS of the cell should be lower, but the cell should still respondby increasing its fractional shortening in the presence of the compound.

H. Additive with Isoproterenol:

To demonstrate that a compound act via a different mechanism than theadrenergic stimulant isoproterenol, cells are loaded with fura-2 andsimultaneous measurement of contractility and calcium ratios aredetermined. The myocytes are sequentially challenged with 5 μm a testcompound, buffer, 2 nM isoproterenol, buffer, and a combination of atest compound and isoproterenol.

Example 7 In Vitro Model of Dose Dependent Cardiac Myosin ATPaseModulation

Bovine and rat cardiac myosins are purified from the respective cardiactissues. Skeletal and smooth muscle myosins used in the specificitystudies are purified from rabbit skeletal muscle and chicken gizzards,respectively. All myosins used in the assays are converted to asingle-headed soluble form (S1) by a limited proteolysis withchymotrypsin. Other sarcomeric components: troponin complex, tropomyosinand actin are purified from bovine hearts (cardiac sarcomere) or chickenpectoral muscle (skeletal sarcomere).

Activity of myosins is monitored by measuring the rates of hydrolysis ofATP. Myosin ATPase is very significantly activated by actin filaments.ATP turnover is detected in a coupled enzymatic assay using pyruvatekinase (PK) and lactate dehydrogenase (LDH). In this assay each ADPproduced as a result of ATP hydrolysis is recycled to ATP by PK with asimultaneous oxidation of NADH molecule by LDH. NADH oxidation can beconveniently monitored by decrease in absorbance at 340 nm wavelength.

Dose responses are measured using a calcium-buffered, pyruvate kinaseand lactate dehydrogenase-coupled ATPase assay containing the followingreagents (concentrations expressed are final assay concentrations):Potassium PIPES (12 mM), MgCl₂ (2 mM), ATP (1 mM), DTT (1 mM), BSA (0.1mg/ml), NADH (0.5 mM), PEP (1.5 mM), pyruvate kinase (4 U/ml), lactatedehydrogenase (8 U/ml), and antifoam (90 ppm). The pH is adjusted to6.80 at 22° C. by addition of potassium hydroxide. Calcium levels arecontrolled by a buffering system containing 0.6 mM EGTA and varyingconcentrations of calcium, to achieve a free calcium concentration of1×10⁻⁴ M to 1×10⁻⁸ M.

The protein components specific to this assay are bovine cardiac myosinsubfragment-1 (typically 0.5 μM), bovine cardiac actin (14 μM), bovinecardiac tropomyosin (typically 3 μM), and bovine cardiac troponin(typically 3-8 μM). The exact concentrations of tropomyosin and troponinare determined empirically, by titration to achieve maximal differencein ATPase activity when measured in the presence of 1 mM EGTA versusthat measured in the presence of 0.2 mM CaCl₂. The exact concentrationof myosin in the assay is also determined empirically, by titration toachieve a desired rate of ATP hydrolysis. This varies between proteinpreparations, due to variations in the fraction of active molecules ineach preparation.

Compound dose responses are typically measured at the calciumconcentration corresponding to 50% of maximal ATPase activity (pCa₅₀),so a preliminary experiment is performed to test the response of theATPase activity to free calcium concentrations in the range of 1×10⁻⁴ Mto 1×10⁻⁸ M. Subsequently, the assay mixture is adjusted to the pCa₅₀(typically 3×10⁻⁷ M). Assays are performed by first preparing a dilutionseries of test compound, each with an assay mixture containing potassiumPipes, MgCl₂, BSA, DTT, pyruvate kinase, lactate dehydrogenase, myosinsubfragment-1, antifoam, EGTA, CaCl₂, and water. The assay is started byadding an equal volume of solution containing potassium Pipes, MgCl₂,BSA, DTT, ATP, NADH, PEP, actin, tropomyosin, troponin, antifoam, andwater. ATP hydrolysis is monitored by absorbance at 340 nm. Theresulting dose response curve is fit by the 4 parameter equationy=Bottom+((Top-Bottom)/(1+((EC50/X)^Hill))). The AC1.4 is defined as theconcentration at which ATPase activity is 1.4-fold higher than thebottom of the dose curve.

Ability of a compound to activate cardiac myosin is evaluated by theeffect of the compound on the actin stimulated ATPase of S1 subfragment.Actin filaments in the assay are decorated with troponin and tropomyosinand Ca++ concentration is adjusted to a value that would result in 50%of maximal activation. S1 ATPase is measured in the presence of adilution series of the compound. Compound concentration required for 40%activation above the ATPase rate measured in the presence of control(equivalent volume of DMSO) is reported as AC₄₀.

Example 8 In Vivo Fractional Shortening Assay

A. Animals

Male Sprague Dawley rats from Charles River Laboratories (275-350 g) areused for bolus efficacy and infusion studies. Heart failure animals aredescribed below. They are housed two per cage and have access to foodand water ad libitum. There is a minimum three-day acclimation periodprior to experiments.

B. Echocardiography

Animals are anesthetized with isoflurane and maintained within asurgical plane throughout the procedure. Core body temperature ismaintained at 37° C. by using a heating pad. Once anesthetized, animalsare shaven and hair remover is applied to remove all traces of fur fromthe chest area. The chest area is further prepped with 70% ETOH andultrasound gel is applied. Using a GE System Vingmed ultrasound system(General Electric Medical Systems), a 10 MHz probe is placed on thechest wall and images are acquired in the short axis view at the levelof the papillary muscles. 2-D M-mode images of the left ventricle aretaken prior to, and after, compound bolus injection or infusion. In vivofractional shortening ((end diastolic diameter−end systolicdiameter)/end diastolic diameter×100) is determined by analysis of theM-mode images using the GE EchoPak software program.

C. Bolus and Infusion Efficacy

For bolus and infusion protocols, fractional shortening is determinedusing echocardiography as described above. For bolus and infusionprotocols, five pre-dose M-Mode images are taken at 30 second intervalsprior to bolus injection or infusion of compounds. After injection,M-mode images are taken at 1 min and at five minute intervals thereafterup to 30 min. Bolus injection (0.5-5 mg/kg) or infusion is via a tailvein catheter. Infusion parameters are determined from pharmacokineticprofiles of the compounds. For infusion, animals received a 1 minuteloading dose immediately followed by a 29 minute infusion dose via atail vein catheter. The loading dose is calculated by determining thetarget concentration×the steady state volume of distribution. Themaintenance dose concentration is determined by taking the targetconcentration×the clearance. Compounds are formulated in 25% cavitronvehicle for bolus and infusion protocols. Blood samples are taken todetermine the plasma concentration of the compounds.

Example 9 Hemodynamics in Normal and Heart Failure Animals

Animals are anesthetized with isoflurane, maintained within a surgicalplane, and then shaven in preparation for catheterization. An incisionis made in the neck region and the right carotid artery cleared andisolated. A 2 French Millar Micro-tip Pressure Catheter (MillarInstruments, Houston, Tex.) is cannulated into the right carotid arteryand threaded past the aorta and into the left ventricle. End diastolicpressure readings, max+/−dp/dt, systolic pressures and heart rate aredetermined continuously while compound or vehicle is infused.Measurements are recorded and analyzed using a PowerLab and the Chart 4software program (ADInstruments, Mountain View, Calif.). Hemodynamicsmeasurements are performed at a select infusion concentration. Bloodsamples are taken to determine the plasma concentration of thecompounds.

Example 10 Left Coronary Artery Occlusion Model of Congestive HeartFailure

A. Animals

Male Sprague-Dawley CD (220-225 g; Charles River) rats are used in thisexperiment. Animals are allowed free access to water and commercialrodent diet under standard laboratory conditions. Room temperature ismaintained at 20-23° C. and room illumination is on a 12/12-hourlight/dark cycle. Animals are acclimatized to the laboratory environment5 to 7 days prior to the study. The animals are fasted overnight priorto surgery.

B. Occlusion Procedure

Animals are anaesthetized with ketamine/xylazine (95 mg/kg and 5 mg/kg)and intubated with a 14-16-gauge modified intravenous catheter.Anesthesia level is checked by toe pinch. Core body temperature ismaintained at 37° C. by using a heating blanket. The surgical area isclipped and scrubbed. The animal is placed in right lateral recumbencyand initially placed on a ventilator with a peak inspiratory pressure of10-15 cm H₂O and respiratory rate 60-110 breaths/min. 100% O₂ isdelivered to the animals by the ventilator. The surgical site isscrubbed with surgical scrub and alcohol. An incision is made over therib cage at the 4^(th)-5^(th) intercostal space. The underlying musclesare dissected with care to avoid the lateral thoracic vein, to exposethe intercostal muscles. The chest cavity is entered through4^(th)-5^(th) intercostal space, and the incision expanded to allowvisualization of the heart. The pericardium is opened to expose theheart. A 6-0 silk suture with a taper needle is passed around the leftcoronary artery near its origin, which lies in contact with the leftmargin of the pulmonary cone, at about 1 mm from the insertion of theleft auricular appendage. The left coronary artery is occluded by tyingthe suture around the artery (“LCO”). Sham animals are treated the same,except that the suture is not tied. The incision is closed in threelayers. The rat is ventilated until able to ventilate on its own. Therats are extubated and allowed to recover on a heating pad. Animalsreceive buprenorphine (0.01-0.05 mg/kg SQ) for post operative analgesia.Once awake, they are returned to their cage. Animals are monitored dailyfor signs of infection or distress. Infected or moribund animals areeuthanized. Animals are weighed once a week.

C. Efficacy Analysis

Approximately eight weeks after infarction surgery, rats are scanned forsigns of myocardial infarction using echocardiography. Only thoseanimals with decreased fractional shortening compared to sham rats areutilized further in efficacy experiments. In all experiments, there arefour groups, sham+vehicle, sham+compound, LCL+vehicle and LCL+compound.At 10-12 weeks post LCL, rats are infused at a select infusionconcentration. As before, five pre-dose M-Mode images are taken at 30second intervals prior to infusion of compounds and M-mode images aretaken at 30 second intervals up to 10 minutes and every minute or atfive minute intervals thereafter. Fractional shortening is determinedfrom the M-mode images. Comparisons between the pre-dose fractionalshortening and compound treatment are performed by ANOVA and a post-hocStudent-Newman-Keuls. Animals are allowed to recover and within 7-10days, animals are again infused with compounds using the hemodynamicprotocol to determine hemodynamic changes of the compounds in heartfailure animals. At the end to the infusion, rats are killed and theheart weights determined.

When tested as described in Examples above, compounds of Formula I areshown to have the desired activity.

Example 11 Cardiac Contractility In Vitro and In Vivo in a Rat Model ofHeart Failure

A myofibril assay is used to identify compounds (myosin activators) thatdirectly activate the cardiac myosin ATPase. The cellular mechanism ofaction, in vivo cardiac function in Sprague Dawley (SD) rats, andefficacy in SD rats with defined heart failure to active compound isthen determined. Cellular contractility was quantified using an edgedetection strategy and calcium transient measured using fura-2 loadedadult rat cardiac myocytes. Cellular contractility increased overbaseline within 5 minutes of exposure to an active compound (0.2 μM)without altering the calcium transient. Combination of active compoundwith isoproterenol (β-adrenergic agonist) should result only in anadditive increase in contractility with no further change in the calciumtransient demonstrating the active compound was not inhibiting the PDEpathway. In vivo contractile function in anesthetized SD rats isquantified using echocardiography (M-mode) and simultaneous pressuremeasurements. SD rats are infused with vehicle or active compound at0.25-2.5 mg/kg/hr. The active compound should increase fractionalshortening (FS) and ejection fraction (EF) in a dose-dependent mannerwith no significant change in peripheral blood pressures or heart rateexcept at the highest dose. Rats with defined heart failure induced byleft coronary ligation, or sham treated rats may have similar andsignificant increases in FS and EF when treated with 0.7-1.2 mg/kg/hractive compound. In summary, the active compound increased cardiaccontractility without increasing the calcium transient and wasefficacious in a rat model of heart failure, indicating the activecompound may be a useful therapeutic in the treatment of human heartfailure.

Example 12 Pharmacology

The pharmacology of at least one chemical entity described herein isinvestigated in isolated adult rat cardiac myocytes, anesthetized rats,and in a chronically instrumented canine model of heart failure inducedby myocardial infarction combined with rapid ventricular pacing. Theactive compound increases cardiac myocyte contractility (EC20=0.2 μM)but does not increase the magnitude or change the kinetics of thecalcium transient at concentrations up to 10 μM in Fura-2 loadedmyocytes. The active compound (30 μM) does not inhibit phosphodiesterasetype 3.

In anesthetized rats, the active compound increases echocardiographicfractional shortening from 45±5.1% to 56±4.6% after a 30 minute infusionat 1.5 mg/kg/hr (n=6, p<0.01).

In conscious dogs with heart failure, the active compound (0.5 mg/kgbolus, then 0.5 mg/kg/hr i.v. for 6-8 hours) increases fractionalshortening by 74±7%, cardiac output by 45±9%, and stroke volume by101±19%. Heart rate decreases by 27±4% and left atrial pressure fallsfrom 22±2 mmHg to 10±2 mmHg (p<0.05 for all). In addition, neither meanarterial pressure nor coronary blood flow changes significantly.Diastolic function is not impaired at this dose. There are nosignificant changes in a vehicle treated group. The active compoundimproved cardiac function in a manner that suggests that compounds ofthis class may be beneficial in patients with heart failure.

Example 13 Pharmaceutical Composition

A pharmaceutical composition for intravenous administration is preparedin the following manner.

1 mg/mL (as free base) IV solution with the vehicle being 50 mM citricacid, pH adjusted to 5.0 with NaOH:

Composition Unit Formula (mg/mL) Active Agent  1.00 Citric Acid 10.51Sodium Hydroxide qs to pH 5.0 Water for Injection (WFI) q.s. to 1 mL*All components other than the active compound are USP/Ph. Eur.compliant

A suitable compounding vessel is filled with WFI to approximately 5% ofthe bulk solution volume. The citric acid (10.51 g) is weighed, added tothe compounding vessel and stirred to produce 1 M citric acid. Theactive agent (1.00 g) is weighed and dissolved in the 1 M citric acidsolution. The resulting solution is transferred to a larger suitablecompounding vessel and WFT is added to approximately 85% of the bulksolution volume. The pH of the bulk solution is measured and adjusted to5.0 with 1 N NaOH. The solution is brought to its final volume (1 liter)with WFI.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. All patents and publications cited above arehereby incorporated by reference.

What is claimed is:
 1. A compound of the formula

or a pharmaceutically acceptable salt thereof, where one of Z¹ and Z² is—NR¹⁵C(O)NR¹⁶R⁴ and the other of Z¹ and Z² is R³; R⁴ is optionallysubstituted heteroaryl; R³ is chosen from hydrogen, halo, cyano,hydroxyl, optionally substituted alkyl, optionally substitutedheterocycloalkyl, and optionally substituted heteroaryl; R¹ and R² areindependently chosen from hydrogen, halo, cyano, optionally substitutedalkyl, optionally substituted heterocycloalkyl, and optionallysubstituted heteroaryl; R¹⁵ and R¹⁶ are independently chosen fromhydrogen, and optionally substituted alkyl; R⁵ is optionally substitutedpiperazinyl; and L is lower alkylene.
 2. A compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R⁵ is chosen from4-(dimethylcarbamoyl)piperazine-1-yl,4-(N,N-dimethylsulfamoyl)piperazine-1-yl, 4-acetyl-piperazin-1-yl,4-ethoxycarbonyl-piperazin-1-yl, 4-ethylsulfonyl-piperazin-1-yl,4-methoxycarbonyl-piperazin-1-yl, 4-methylsulfonyl-piperazin-1-yl,4-t-butoxycarbonyl-piperazin-1-yl, piperazin-1-yl,4-(4-acetylpiperazine-1-carbonyl)piperazin-1-yl,4-(4-methylpiperazine-1-carbonyl)piperazin-1-yl,4-(piperidine-1-carbonyl)piperazin-1-yl,4-(morpholine-4-carbonyl)piperazin-1-yl,4-(cyclobutylsulfonyl)-piperazin-1-yl, 4-(ethylsulfonyl)piperazin-1-yl,4-(isopropylsulfonyl)piperazin-1-yl,4-(cyclopropylsulfonyl)piperazin-1-yl, and 4-(1,1-dioxidethiomorpholine-4-carbonyl)piperazin-1-yl.
 3. A compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R⁴ is chosen fromoptionally substituted pyridinyl.
 4. A compound of claim 3, or apharmaceutically acceptable salt thereof, wherein R⁴ is chosen from6-methoxy-pyridin-3-yl, 6-methyl-pyridin-3-yl and pyridin-3-yl.
 5. Acompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R³ is chosen from hydrogen, halo, cyano, lower alkyl, andhydroxyl.
 6. A compound of claim 5, or a pharmaceutically acceptablesalt thereof, wherein R³ is chosen from hydrogen, fluoro, chloro,methyl, ethyl and hydroxyl.
 7. A compound of claim 6, or apharmaceutically acceptable salt thereof, wherein R¹ and R² areindependently chosen from hydrogen, halo, cyano and lower alkyl.
 8. Acompound of claim 1, or a pharmaceutically acceptable salt thereof whereR¹, R² and R³ are hydrogen.